Preparation method and application of engineered cell vesicle nano vaccine for enhancing cancer immunotherapy

Abstract

The invention provides a preparation method and application of an engineered cell vesicle nano vaccine for enhancing cancer immunotherapy, and belongs to the technical field of biological medicines.The preparation method comprises the steps that tumor cells subjected to heat shock treatment are mechanically extruded, and TEV is obtained; dissolving sodium chloride, hydrochloric acid and tetramethoxysilane in water to obtain a silicic acid solution, then adding the TEV, and incubating to obtain Si (at) TEV; the preparation method comprises the following steps: dissolving polyethyleneimine in an organic solvent, then adding a fluorinating reagent 3-perfluorohexyl-1, 2-epoxypropane, and carrying out stirring reaction, dialysis and freeze-drying to obtain F-PEI; the F-PEI is dissolved in PBS, then the Si (at) TEV is added, and the nano vaccine F-PEI / Si (at) TEV is obtained through stirring reaction, washing and centrifugation. The engineered cell vesicle nano vaccine prepared by the method is good in stability, can effectively improve the anti-tumor immune response of an organism and powerfully kill tumor cells, and can be applied to prevention and treatment of different types of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a preparation method and application of an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy. Background Art

[0002] Tumor vaccines are an immunotherapy strategy that utilizes tumor-specific or tumor-associated antigens (such as tumor cells, tumor-derived vesicles, tumor-associated proteins or peptides, etc.) in combination with adjuvants to interact with antigen-presenting cells (APCs) to activate the body's specific immune response and kill tumor cells. They are a hot topic in tumor immunotherapy research. However, traditional tumor vaccines still face many challenges in practical application, such as insufficient immunogenicity, lack of interaction with APCs, and low antigen presentation efficiency, which seriously hinder the clinical translation and application of tumor vaccines. Therefore, there is an urgent need to develop new delivery systems to enhance vaccine targeting, optimize adjuvant formulations to improve immune activation efficiency, and combine emerging technologies to design more efficient personalized tumor vaccines. Summary of the Invention

[0003] In order to obtain a new tumor vaccine, the present invention provides a method for preparing an engineered cell vesicle nanovaccine that enhances cancer immunotherapy. The engineered cell vesicle nanovaccine prepared by this method has good stability, can effectively enhance the body's anti-tumor immune response, and potently kill tumor cells, and can be applied to the prevention and treatment of different types of tumors.

[0004] The present invention also provides an engineered cell vesicle nano vaccine for enhancing cancer immunotherapy and its application.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention provides a method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy, the preparation method comprising:

[0007] Mechanically squeeze heat-shocked tumor cells to obtain TEVs;

[0008] Sodium chloride, hydrochloric acid and tetramethoxysilane are dissolved in water to obtain a silicic acid solution, and then the TEV is added and incubated to obtain Si@TEV;

[0009] Polyethyleneimine is dissolved in an organic solvent, and then a fluorination reagent 3-perfluorohexyl-1,2-propylene oxide is added. After stirring, reaction, dialysis and freeze-drying, F-PEI is obtained.

[0010] The F-PEI was dissolved in PBS, and then the Si@TEV was added. After stirring, reaction, washing and centrifugation, the nanovaccine F-PEI / Si@TEV was obtained.

[0011] Furthermore, the mechanical extrusion of the heat-shocked tumor cells to obtain TEVs specifically includes:

[0012] Tumor cells were placed at 43 ± 0.5°C for heat shock treatment to obtain heat shock cells;

[0013] The heat-shocked cells were resuspended in PBS, and the resuspended solution was mechanically squeezed through 0.8 μm, 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain a PBS suspension of TEV;

[0014] The TEV suspension in PBS was centrifuged at 3-5° C. to collect the TEV.

[0015] Furthermore, the sodium chloride, hydrochloric acid and tetramethoxysilane are dissolved in water to obtain a silicic acid solution, and then the TEV is added and incubated to obtain Si@TEV, which specifically includes:

[0016] dissolving sodium chloride, hydrochloric acid and tetramethoxysilane in water to obtain a silicic acid solution;

[0017] adding the TEV to the silicic acid solution, incubating at room temperature and then incubating at -80±1° C. to obtain a siliconized solution;

[0018] The siliconized solution was washed with PBS and resuspended to obtain Si@TEV;

[0019] Wherein, in the silicic acid solution, the molar ratio of sodium chloride, HCl and tetramethoxysilane is 154:1:100;

[0020] The concentration of sodium chloride in the silicic acid solution is 100-154 mM.

[0021] Furthermore, the method of dissolving polyethyleneimine in an organic solvent, adding a fluorination reagent 3-perfluorohexyl-1,2-propylene oxide, stirring, reacting, dialysis and freeze-drying to obtain F-PEI specifically includes:

[0022] Dissolve polyethyleneimine in methanol, then add the fluorination reagent 3-perfluorohexyl-1,2-propylene oxide, stir at room temperature for 48±1h, then dialyze with a 1000Da dialysis bag for 72±1h, and lyophilize to obtain F-PEI;

[0023] The molar ratio of polyethyleneimine to 3-perfluorohexyl-1,2-propylene oxide is 1:(25-100).

[0024] Furthermore, the F-PEI is dissolved in PBS, and then the Si@TEV is added, stirred, reacted, washed and centrifuged to obtain the nanovaccine F-PEI / Si@TEV, which specifically includes:

[0025] The F-PEI was dissolved in PBS and then added to the Si@TEV, stirred at room temperature for 20±5 min, washed with PBS, and then centrifuged at 3-5°C to obtain the nanovaccine F-PEI / Si@TEV.

[0026] Preferably, the tumor cells include at least one of mouse tumor cells B16F10, breast cancer cells, colon cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells and melanoma cells.

[0027] Based on the same inventive concept, the present invention provides an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy, wherein the nanovaccine is prepared by the above-mentioned method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy;

[0028] or:

[0029] The nanovaccine comprises an immune adjuvant and an antigen. The immune adjuvant is modified on the surface of the antigen through electrostatic interaction. The immune adjuvant is prepared by amine-epoxide reaction between polyvinylamine and 3-perfluorohexyl-1,2-propylene oxide. The antigen is prepared by low-temperature silicification of tumor-derived cell vesicles treated with heat shock.

[0030] Based on the same inventive concept, the present invention provides an application of an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy in the preparation of tumor prevention and treatment drugs.

[0031] Furthermore, the tumor includes at least one of breast cancer, colon cancer, lung cancer, liver cancer, gastric cancer and melanoma.

[0032] Based on the same inventive concept, the present invention also provides a drug for preventing or treating tumors, wherein the active ingredient of the drug includes the above-mentioned engineered cell vesicle nanovaccine for enhancing cancer immunotherapy.

[0033] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0034] 1. The present invention discloses a method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy. The method first mechanically squeezes heat-shocked tumor cells to obtain highly immunogenic tumor cell vesicles (TEVs). Then, a nano-silicon dioxide layer is coated on the surface of the TEV using low-temperature silicification technology to construct a silicified tumor cell vesicle (Si@TEV), significantly improving the mechanical stiffness and stability of the TEV. Finally, the fluorination reagent 3-perfluorohexyl-1,2-propylene oxide is used to modify polyethyleneimine to generate a hydrophobic and lipophobic immune adjuvant, fluorinated polyethyleneimine (F-PEI), which is then electrostatically adsorbed. Modified on the Si@TEV surface, F-PEI / Si@TEV nanovaccine is constructed. The nanovaccine prepared by this method can effectively activate antigen-presenting cells, promote the maturation of antigen-presenting cells, and induce tumor antigen-specific T cell immunity, thereby effectively killing tumor cells, enhancing the body's anti-tumor immune response, and inducing tumor cell-specific damage. This method is simple and easy to produce on a large scale. The silicified cell vesicles from tumor cells can adapt to dehydration and room temperature storage without losing efficacy. They are highly stable and have good universality, which helps to prepare personalized cancer vaccines across tumor types and can be used in the prevention and treatment of different types of tumors.

[0035] 2. The present invention discloses an engineered cell vesicle nano-vaccine for enhancing cancer immunotherapy. The nano-vaccine is prepared by mechanically extracting highly immunogenic TEV from heat-shocked tumor cells and coating the surface with a layer of nano-silica using low-temperature silanization technology to obtain a high-quality antigen portion Si@TEV. The antigen portion is then synthesized with polyethyleneimine and a fluorination reagent, 3-perfluorohexyl-1,2-propylene oxide (F 13 ) The immune adjuvant F-PEI with hydrophobic and lipophobic properties was synthesized by chemical reaction and modified with the Si@TEV surface through classical interactions to form the cell vesicle nanovaccine F-PEI / Si@TEV. F-PEI / Si@TEV enhances the body's immune activation efficiency through three key cascade pathways: 1) TEV after heat shock treatment enhances the expression of antigens such as heat shock protein 70 (HSP70), thereby improving the immunogenicity of tumor vaccines; 2) Si@TEV after low-temperature siliconization has high mechanical stiffness, which enables the vaccine to activate the Hippo-YAP / TAZ pathway of DCs through mechanical signal transduction, inducing stronger DCs phagocytic effect and migration ability; 3) F-PEI / Si@TEV nanovaccine activates TLR and NF-κB signaling pathways in DCs through the immune adjuvant F-PEI, promoting DCs activation and antigen presentation efficiency. The three work together to form an efficient immune activation network, significantly enhancing the body's ability to recognize and attack tumor cells, and ultimately achieving efficient inhibition and long-term control of tumors.

[0036] 3. The present invention discloses an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy. The F-PEI / Si@TEV nanovaccine has a uniform particle size of 234.3 nm, which can increase the phagocytosis and antigen presentation efficiency of the vaccine by antigen-presenting cells, thereby achieving excellent immune activation. F-PEI / Si@TEV has good stability and can be stored for a long time at 4°C. The nanovaccine F-PEI / Si@TEV vaccine prepared by the present invention significantly improves the anti-tumor immune efficacy through the synergistic effect of multiple mechanisms, exerts a powerful immune activation effect in vivo and in vitro, and thus enhances the anti-tumor immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 Schematic diagram of the synthesis of the nanovaccine F-PEI / Si@TEV of the present invention.

[0039] Figure 2 This is the elemental analysis (mapping) diagram of Si@TEV.

[0040] Figure 3 F 13 and NMR fluorine spectra of F-PEI.

[0041] Figure 4 This is the transmission electron microscopy (TEM) image of F-PEI / Si@TEV.

[0042] Figure 5 This is the atomic force microscopy (AFM) image of F-PEI / Si@TEV.

[0043] Figure 6 Young's modulus diagram of each material.

[0044] Figure 7 These are the results of the cytotoxicity experiment of the nanovaccine.

[0045] Figure 8 The results of antigen-presenting cell uptake of each substance.

[0046] Figure 9 Immune cell recruitment results for each substance.

[0047] Figure 10 The ability of each substance to activate immunity in vitro. DETAILED DESCRIPTION

[0048] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0049] Throughout this specification, unless otherwise specified, the terms used herein should be understood as having the same meaning as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0050] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] The overall concept of the present invention is as follows:

[0053] Traditional vaccine design faces three core challenges: insufficient antigen immunogenicity, making it difficult to effectively activate naive T cells; low antigen presentation efficiency, leading to lysosomal degradation; and the immunosuppressive nature of the tumor microenvironment, which weakens vaccine efficacy. Therefore, the key breakthrough in tumor vaccine development lies in how to build efficient delivery systems and synergistically regulate the immune microenvironment through cross-innovation in materials science and immunology.

[0054] Extracellular vesicles (EVs), as an important medium for intercellular communication, naturally carry the proteins, nucleic acids, and lipid components of the source cells. They have the advantages of high biocompatibility and natural targeting, and are an excellent natural bio-nanomaterial. The inventors have found that tumor-derived extracellular vesicles (TEVs) are not only rich in tumor-specific antigens, but can also mediate homologous tumor targeting through surface adhesion molecules such as cadherins. At the same time, TEVs have good biocompatibility and are almost non-toxic, providing an ideal antigen library and delivery platform for vaccine design. Therefore, the inventors believe that by using technologies such as low-temperature siliconization and chemical modification to perform multiple engineering modifications on cellular vesicles, it is expected that stable TEVs can be quickly prepared, protected from environmental stress, and the immune activation ability of TEVs can be enhanced, which is expected to promote breakthrough progress in tumor vaccine technology.

[0055] Based on this, the present invention provides an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy and its preparation method. The nanovaccine is prepared by extracting highly immunogenic TEV from heat-shocked tumor cells through mechanical extrusion, and coating the surface of the cells with a layer of nano-silica using low-temperature silanization technology to obtain a high-quality antigen portion Si@TEV. The antigen is then synthesized with polyethyleneimine and a fluorination reagent 3-perfluorohexyl-1,2-propylene oxide (F 13 ) The hydrophobic and lipophobic immune adjuvant F-PEI was synthesized through chemical reactions and then modified with the Si@TEV surface through classical interactions to form the cell vesicle nanovaccine F-PEI / Si@TEV. F-PEI / Si@TEV enhances the body's immune activation efficiency through three key cascade pathways: 1) Heat-shocked TEVs enhance the expression of antigens such as heat shock protein 70 (HSP70), improving the immunogenicity of tumor vaccines; 2) Si@TEVs after low-temperature siliconization have high mechanical stiffness, allowing the vaccine to activate the Hippo-YAP / TAZ pathway in DCs through mechanical signal transduction, inducing stronger DC phagocytosis and migration; 3) The F-PEI / Si@TEV nanovaccine activates signaling pathways such as TLR and NF-κB in DCs through the immune adjuvant F-PEI, promoting DC activation and antigen presentation efficiency. The three synergistically form a highly efficient immune activation network, significantly enhancing the body's ability to recognize and attack tumor cells, ultimately achieving efficient inhibition and long-term control of tumors.

[0056] Mechanical signals can be converted into biochemical signals through the mechanical transduction mechanism, thereby regulating various biological behaviors of immune cells. There are a variety of mechanically sensitive signaling pathways in immune cells, which can significantly affect key functions such as cell activation, cytokine secretion, metabolic reprogramming, proliferation and migration. Among them, as the core antigen-presenting cells connecting innate immunity and adaptive immunity, the functional state of dendritic cells (DCs) is precisely regulated by the mechanical microenvironment. The inventors found that matrix stiffness can affect the metabolic characteristics, phenotypic differentiation and inflammatory function of DCs by regulating the activity of downstream effector molecules of the Hippo signaling pathway, which suggests that mechanical force stimulation plays an important role in DCs metabolic reprogramming, phagocytic activation and immune function regulation. Based on this, the present invention designs a biomaterial system with adjustable stiffness for optimizing DCs mechanical transduction pathways. Quantitative characterization by atomic force microscopy showed that the Young's modulus of the simple vesicles was 2.27 MPa; after being coated on the surface of nano-silica, the Young's modulus of the siliconized vesicles was significantly increased to 11.29 MPa; after further modification of the immune adjuvant F-PEI, the Young's modulus of the composite vaccine was maintained at 10.61 MPa. It is worth noting that the adjuvant modification did not significantly change the mechanical properties of the material, which indicates that the low-temperature siliconization process can effectively enhance the mechanical stiffness of the vaccine. This stiffness value is significantly higher than the physiological environment level, so that when the vaccine acts on DCs, in addition to exerting the activation effect of chemical adjuvants, it can also activate the Hippo-YAP / TAZ signaling pathway through mechanical force stimulation: on the one hand, it promotes the activation of DCs, and on the other hand, it enhances its antigen uptake ability and migration efficiency by inducing DCs cytoskeleton reorganization, thereby synergistically enhancing the anti-tumor immune response.

[0057] The cell vesicle nanovaccine designed in the present invention can be targeted to the body's lymph nodes through lymphatic drainage vessels, and combined with the TLR4 ligand on the surface of DCs through F-PEI, so that the vaccine can be specifically taken up by DCs and directly interact with DCs. The enhanced mechanical stiffness of the vaccine enables it to activate the Hippo-YAP / TAZ signaling pathway of DCs through mechanical signal transduction, promote DCs activation, and induce stronger DCs phagocytic effect and migration ability.

[0058] Specifically, the present invention provides a method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy, the preparation method comprising:

[0059] S1. Mechanically squeeze heat-shocked tumor cells to obtain TEVs.

[0060] S2. Sodium chloride, hydrochloric acid and tetramethoxysilane were dissolved in water to obtain a silicic acid solution, and then the TEV was added and incubated to obtain Si@TEV;

[0061] S3. Polyethyleneimine was dissolved in an organic solvent, and then a fluorination reagent 3-perfluorohexyl-1,2-propylene oxide was added. The mixture was stirred, dialyzed, and freeze-dried to obtain F-PEI.

[0062] S4. The F-PEI was dissolved in PBS, and then the Si@TEV was added. After stirring, reaction, washing and centrifugation, the nanovaccine F-PEI / Si@TEV was obtained.

[0063] Step S1 specifically includes:

[0064] Tumor cells were placed at 43 ± 0.5°C for heat shock treatment to obtain heat shock cells;

[0065] The heat-shocked cells were resuspended in PBS, and the resuspended solution was mechanically squeezed through 0.8 μm, 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain a PBS suspension of TEV;

[0066] The TEV suspension in PBS was centrifuged at 3-5° C. to collect the TEV.

[0067] In the present invention, heat shock treatment of tumor cells can increase the expression level of antigens such as heat shock protein 70 (HSP70) in the cells, thereby extracting highly immunogenic TEV and improving the immunogenicity of subsequent vaccines.

[0068] In this study, mechanically extruding heat-shocked tumor cells to extract TEVs significantly increases TEV yield and facilitates large-scale production. Compared to collecting vesicles secreted by tumor cells themselves, this method effectively retains the abundant tumor antigens present in the parent cells.

[0069] Step S2 specifically includes:

[0070] dissolving sodium chloride, hydrochloric acid and tetramethoxysilane in water to obtain a silicic acid solution;

[0071] adding the TEV to the silicic acid solution, incubating at room temperature and then incubating at -80±1° C. to obtain a siliconized solution;

[0072] The siliconized solution was washed with PBS and resuspended to obtain Si@TEV;

[0073] Wherein, in the silicic acid solution, the molar ratio of sodium chloride, HCl and tetramethoxysilane is 154:1:100;

[0074] The concentration of sodium chloride in the silicic acid solution is 100-154 mM.

[0075] In the present invention, low-temperature siliconization treatment of TEV can give TEV greater mechanical stiffness, protecting it from environmental stress. At the same time, the vaccine activates the Hippo-YAP / TAZ pathway of dendritic cells (DCs) through mechanical signal transduction, inducing stronger DCs phagocytic effect and migration ability.

[0076] In the present invention, the molar ratio of sodium chloride, HCl and tetramethoxysilane is 154:1:100. The Si@TEV prepared with this ratio has good stability and dispersibility.

[0077] In the present invention, TEV is added to the silicic acid solution and incubated at room temperature before being placed at -80±1°C. The advantage is that the nano-silica layer deposited on the surface of Si@TEV is thin, allowing rapid dissolution of silica without affecting the biomolecular functions of the TEV component.

[0078] Step S3 specifically includes:

[0079] Dissolve polyethyleneimine in methanol, then add the fluorination reagent 3-perfluorohexyl-1,2-propylene oxide, stir at room temperature for 48±1h, then dialyze with a 1000Da dialysis bag for 72±1h, and lyophilize to obtain F-PEI;

[0080] The molar ratio of polyethyleneimine to 3-perfluorohexyl-1,2-propylene oxide is 1:(25-100).

[0081] In this study, polyethylenimine was modified with the fluorinated reagent 3-perfluorohexyl-1,2-propylene oxide, addressing the bottlenecks of traditional polyethylenimine adjuvants, such as low cellular uptake efficiency and high cytotoxicity. The introduction of a fluorinated ligand not only enhances the stability of the adjuvant-antigen complex by leveraging its hydrophobic and lipophobic properties, but also achieves superior immune activation and more precise intracellular delivery by regulating the interaction dynamics between the carrier and the biomembrane.

[0082] In the present invention, the molar ratio of polyethyleneimine to 3-perfluorohexyl-1,2-propylene oxide is 1:(25-100). The fluorinated polyethyleneamine synthesized at this ratio has the most excellent immune activation effect.

[0083] Step S4 specifically includes:

[0084] The F-PEI was dissolved in PBS and then added to the Si@TEV, stirred at room temperature for 20±5 minutes, washed with PBS, and then centrifuged at 3-5°C to obtain the nanovaccine F-PEI / Si@TEV;

[0085] The mass ratio of the F-PEI to the Si@TEV is (0.5-3):1.

[0086] In the present invention, Si@TEV is surface-modified with F-PEI. On the one hand, the immune adjuvant F-PEI modified on the vaccine surface directly stimulates the activation of DCs in the immune system. On the other hand, low-temperature siliconization treatment is used to impart higher mechanical stiffness to the vaccine, providing additional mechanical stimulation through mechanical transduction signals, further activating the immune system and thus exerting a powerful anti-tumor immune response.

[0087] The advantage of the mass ratio of F-PEI to Si@TEV in the present invention being (0.5-3):1 is that the nanovaccine F-PEI / Si@TEV synthesized in this ratio has the best immune activation effect.

[0088] The following is a detailed description of the preparation method and application of an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy according to the present invention, in combination with examples and experimental data.

[0089] Example 1

[0090] like Figure 1 As shown, this embodiment provides a method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy, as follows:

[0091] (1) Preparation of antigen part Si@TEV:

[0092] TEV extraction: Mouse melanoma cell line B16F10 was cultured in RPMI 1640 cell culture medium. When cells were confluent, they were heat-shocked in a 43°C water bath for 2 h. After the cells were digested with trypsin, the cell pellet was collected, washed with PBS, and resuspended. The cell-PBS suspension was aspirated with a syringe and mechanically squeezed through 0.8, 0.45, and 0.22 μm microporous membrane filters. The collected PBS solution was centrifuged at 10,000 g for 10 min at 4°C to remove debris. The supernatant was centrifuged again at 120,000 g for 2 h at 4°C to obtain the TEV precipitate. The TEV precipitate was washed twice and resuspended in PBS for subsequent experiments.

[0093] Preparation of silicic acid solution: dissolve 5.4 mg of sodium chloride, 1.5 μL of tetramethoxysilane, and 83.3 μL of 1000-fold diluted concentrated hydrochloric acid (concentrated hydrochloric acid concentration 12M-13M) in deionized water to prepare 1 mL of silicic acid solution.

[0094] Preparation of Si@TEV: 100 μg of TEV (TEV resuspension) was added to 1 mL of silicic acid solution and incubated at room temperature for 10 minutes. The mixture was then transferred to a -80°C freezer and incubated for another 24 hours. After silanization, the silicic acid solution was removed by centrifugation. The cells were washed twice with deionized water and then PBS. The supernatant was discarded and the pellet was resuspended in fresh PBS to obtain Si@TEV.

[0095] (2) Preparation of immune adjuvant F-PEI: 10 mg of PEI was accurately weighed and dissolved in 2 mL of anhydrous methanol, and then the fluorination reagent F was added at a molar ratio of 1:50. 13 The solution was added dropwise to a methanol solution of PEI and stirred at room temperature for 48 hours. After the reaction was complete, the solution was transferred to a 1000Da dialysis bag and dialyzed in deionized water for 72 hours. The liquid at the end of the dialysis was collected and freeze-dried to obtain the immune adjuvant F-PEI.

[0096] (3) Preparation of nanovaccine F-PEI / Si@TEV: Accurately weigh 0.2 mg of F-PEI and dissolve it in 1 mL of PBS. Then, add the Si@TEV resuspension to the PBS solution at a mass ratio of 2:1. Stir at room temperature for 20 min. After sufficient reaction, wash twice with PBS to remove free F-PEI. Centrifuge at 4°C and 120,000 g for 2 h to obtain the nanovaccine F-PEI / Si@TEV.

[0097] Example 2

[0098] In this example, the nanovaccine obtained in Example 1 was subjected to functional tests, as follows:

[0099] (1) Elemental Analysis (Mapping)

[0100] Take 10μL of Si@TEV solution of appropriate concentration and carefully drip it onto the surface of the copper mesh. Let it stand for 5 minutes, absorb the excess solution with filter paper strips, and after it is completely dried, use the mapping function attached to the TEM to analyze the element types of Si@TEV.

[0101] The results of the test are as follows Figure 2 The surface of Si@TEV has a uniform distribution of C, O, and Si elements, which proves that the surface of TEV is covered with a nano-silica layer and Si@TEV is successfully prepared.

[0102] (2) H NMR spectroscopy ( 19 F NMR)

[0103] Take appropriate amount of F 13 , F-PEI, was fully dissolved in 1 mL of deuterated methanol (CD3OD), transferred to a nuclear magnetic resonance sample tube and tested on the machine, where the test frequency of the nuclear magnetic resonance spectrometer was 376.48 MHz.

[0104] The results of the test are as follows Figure 3 , belongs to F 13The characteristic peaks of fluorine atoms, including d = -82.41ppm (3F, -CF3), -113.80ppm (2F, -CH2-CF2-), -124.57~-122.77ppm (6F, -CF2-CF2-CF2-), and -127.31ppm (2F, -CF2-CF3), all appeared in the NMR fluorine spectrum of F-PEI, further proving the successful preparation of F-PEI.

[0105] (3) Transmission electron microscopy (TEM) images

[0106] Take a F-PEI / Si@TEV solution of appropriate concentration, add 10 μL onto the surface of the copper mesh, let it stand for 5 minutes, absorb the excess solution with a filter paper strip, wait for the sample to completely dry at room temperature, and then use TEM to observe the sample and record the image.

[0107] The results of the test are as follows Figure 4 ,The surface of F-PEI / Si@TEV vaccine is coated with a layer of dendritic material, which is uniform in size and has a particle size of about 230nm.

[0108] (4) Atomic force microscopy (AFM) images

[0109] Take a F-PEI / Si@TEV solution of appropriate concentration, drop 10 μL on the surface of the mica sheet, place it on the AFM platform for observation, and record the sample morphology.

[0110] The results of the test are as follows Figure 5 , the F-PEI / Si@TEV vaccine is evenly distributed and uniform in size.

[0111] (5) Determination of Young's modulus of nanovaccine

[0112] Take a F-PEI / Si@TEV solution of appropriate concentration, drop 10 μL on the surface of the mica sheet, place it on the AFM platform for observation, and record the Young's modulus of the sample.

[0113] The results of the test are as follows Figure 6 The mechanical stiffness of pure TEV is poor, with a Young's modulus of about 2.27 MPa. After low-temperature silicification treatment, the mechanical stiffness of Si@TEV is significantly enhanced, and the Young's modulus increases to 11.29 MPa. After its surface is modified with F-PEI, the Young's modulus of F-PEI / Si@TEV is slightly reduced to about 10.61 MPa, but there is no significant difference in the Young's modulus of Si@TEV, and it does not affect the overall mechanical stiffness of the preparation.

[0114] (6) Biosafety assessment of nanovaccines

[0115] DC2.4 and RAW264.7 cells in good condition were cultured at a rate of 1×104 / well were seeded into 96-well plates. After culturing in the incubator for 24 hours, the original culture medium was discarded, and 200 μL of cell culture medium containing different concentrations of F-PEI / Si@TEV (drug concentration gradient of 0, 5, 10, 15, 20, 15 μg / mL, culture medium was 1640 culture medium containing 10% fetal bovine serum) was added to each well, with 6 parallel wells set for each concentration. After incubation in the incubator for another 24 hours, the drug-containing culture medium was discarded, and fresh culture medium containing CCK-8 working solution was added to make each well contain 10 μL CCK-8 working solution. After incubation in the incubator for another 2 hours in the dark, the absorbance of each group was detected at a wavelength of 450 nm using a microplate reader, and the cell survival rate of each group was calculated.

[0116] The results are as follows Figure 7 As shown, compared with the control group, the survival rates of the two immune cells treated with different concentrations of the preparations after a certain period of culture were all above 80%. This result shows that FPEI / Si@TEV has no significant effect on cell growth within the tested concentration range and does not show obvious cytotoxicity, showing good biocompatibility and safety.

[0117] (7) Uptake of Nanovaccines by Antigen-Presenting Cells

[0118] The extracted BMDCs were divided into 5×10 5 The cells were seeded into 6-well plates at a density of 100 cells / well and cultured in an incubator for 24 hours. TEV was labeled with DiO dye, and DiO-labeled Si@TEV and F-PEI / Si@TEV were prepared using the TEV. When the cell density in the 6-well plate reached about 70%, the old culture medium was discarded and different fresh drug-containing culture media were added (drug concentration was 3 μg / mL, and the culture medium was 1640 medium containing 10% fetal bovine serum). The 6-well plate was continued to be incubated in the incubator in the dark for 6 hours, washed three times with PBS, and the cell uptake was examined by flow cytometry.

[0119] The results are as follows Figure 8 Compared to unmodified TEV, the nano-silica-coated Si@TEV exhibited enhanced cellular uptake due to a significant increase in material stiffness. This phenomenon can be attributed to the nano-silica coating altering the physical properties of the formulation, making it more easily recognized and taken up by BMDCs. Furthermore, the Si@TEV surface was modified with F-PEI. Due to F-PEI's unique hydrophobic and lipophobic properties, the uptake efficiency of the formulation by BMDCs was further enhanced.

[0120] (8) Immune cell recruitment ability of each substance

[0121] BMDCs were cultured at a rate of 5 × 10 4BMDCs were seeded at a density of 100 cells / well into the upper chamber of a transwell with a pore size of 5 μm. The lower chamber was filled with culture medium containing PEI, F-PEI, TEV, Si@TEV, F-PEI / TEV, and F-PEI / Si@TEV (drug concentration: 3 μg / mL, culture medium: 1640 medium supplemented with 10% fetal bovine serum), respectively. A control group was incubated with blank culture medium alone. After 24 or 48 hours of co-incubation, the number of BMDCs migrating into the lower chamber was observed under a microscope and recorded.

[0122] The results are as follows Figure 9 Compared with the control group, at 24 h, the number of BMDCs cells migrating to the transwell chamber in each experimental group increased to a certain extent. At 48 h, the recruitment effect of all experimental groups was improved compared with that at 24 h. Among them, the final preparation F-PEI / Si@TEV group always showed the strongest recruitment ability, and the cell density in the transwell chamber was much higher than that of other groups.

[0123] (9) The ability of each substance to activate immunity in vitro.

[0124] BMDCs in good condition were cultured at a rate of 5×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate. After 24 hours of culture, the old culture medium was discarded and culture medium containing PEI, F-PEI, TEV, Si@TEV, F-PEI / TEV, and F-PEI / Si@TEV (drug concentration of 3 μg / mL, culture medium 1640 supplemented with 10% fetal bovine serum) was added and incubated for another 24 hours. A control group was treated with blank culture medium only. After incubation, the cell pellet was treated with BB700 anti-mouse CD11c, BV421 anti-mouse CD80, and PE-CY7 anti-mouse CD86 antibodies, respectively, and stained at 4°C for 30 minutes. After washing twice with PBS, the cell pellet was collected and the expression of CD80 and CD86 on the cells was analyzed by flow cytometry.

[0125] The results are as follows Figure 10 The simple TEV antigen showed a weak immune activation effect, but after low-temperature siliconization treatment and F-PEI modification, the ability of the vaccine to stimulate BMDCs maturation was significantly enhanced. F-PEI / Si@TEV showed excellent immune activation effect in in vitro experiments.

[0126] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0127] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0128] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy, characterized in that: The preparation method comprises: Mechanically squeeze heat-shocked tumor cells to obtain TEVs; Sodium chloride, hydrochloric acid and tetramethoxysilane are dissolved in water to obtain a silicic acid solution, and then the TEV is added and incubated to obtain Si@TEV; Polyethyleneimine is dissolved in an organic solvent, and then a fluorination reagent 3-perfluorohexyl-1,2-propylene oxide is added. After stirring, reaction, dialysis and freeze-drying, F-PEI is obtained. The F-PEI was dissolved in PBS, and then the Si@TEV was added. After stirring, reaction, washing and centrifugation, the nanovaccine F-PEI / Si@TEV was obtained.

2. The method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy according to claim 1, characterized in that: The mechanical extrusion of the heat-shocked tumor cells to obtain TEVs specifically includes: Tumor cells were placed at 43 ± 0.5°C for heat shock treatment to obtain heat shock cells; The heat-shocked cells were resuspended in PBS, and the resuspended solution was mechanically squeezed through 0.8 μm, 0.45 μm and 0.22 μm microporous filter membranes in sequence to obtain a PBS suspension of TEV; The TEV suspension in PBS was centrifuged at 3-5° C. to collect the TEV.

3. The method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy according to claim 1, characterized in that: The step of dissolving sodium chloride, hydrochloric acid and tetramethoxysilane in water to obtain a silicic acid solution, adding the TEV, and incubating to obtain Si@TEV specifically comprises: dissolving sodium chloride, hydrochloric acid and tetramethoxysilane in water to obtain a silicic acid solution; adding the TEV to the silicic acid solution, incubating at room temperature and then incubating at -80±1° C. to obtain a siliconized solution; The siliconized solution was washed with PBS and resuspended to obtain Si@TEV; Wherein, in the silicic acid solution, the molar ratio of sodium chloride, HCl and tetramethoxysilane is 154:1:100; The concentration of sodium chloride in the silicic acid solution is 100-154 mM.

4. The method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy according to claim 1, characterized in that: The method comprises dissolving polyethyleneimine in an organic solvent, adding a fluorination reagent 3-perfluorohexyl-1,2-propylene oxide, reacting by stirring, dialysis and freeze-drying to obtain F-PEI, which specifically comprises: Dissolve polyethyleneimine in methanol, then add the fluorination reagent 3-perfluorohexyl-1,2-propylene oxide, stir at room temperature for 48±1h, then dialyze with a 1000Da dialysis bag for 72±1h, and lyophilize to obtain F-PEI; The molar ratio of polyethyleneimine to 3-perfluorohexyl-1,2-propylene oxide is 1:(25-100).

5. The method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy according to claim 1, characterized in that: The F-PEI is dissolved in PBS, and then the Si@TEV is added, stirred for reaction, washed and centrifuged to obtain the nano vaccine F-PEI / Si@TEV, which specifically includes: The F-PEI was dissolved in PBS and then added to the Si@TEV, stirred at room temperature for 20±5 min, washed with PBS, and then centrifuged at 3-5°C to obtain the nanovaccine F-PEI / Si@TEV.

6. The method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy according to claim 1, characterized in that: The tumor cells include at least one of mouse tumor cells B16F10, breast cancer cells, colon cancer cells, lung cancer cells, liver cancer cells, gastric cancer cells and melanoma cells.

7. An engineered cell vesicle nanovaccine for enhancing cancer immunotherapy, characterized in that: The nanovaccine is prepared by the method for preparing an engineered cell vesicle nanovaccine for enhancing cancer immunotherapy according to any one of claims 1 to 6; or: The nanovaccine comprises an immune adjuvant and an antigen. The immune adjuvant is modified on the surface of the antigen through electrostatic interaction. The immune adjuvant is prepared by amine-epoxide reaction between polyvinylamine and 3-perfluorohexyl-1,2-propylene oxide. The antigen is prepared by low-temperature silicification of tumor-derived cell vesicles treated with heat shock.

8. Use of the engineered cell vesicle nanovaccine for enhancing cancer immunotherapy as claimed in claim 7 in the preparation of tumor prevention and treatment drugs.

9. The use according to claim 8, characterized in that The tumor includes at least one of breast cancer, colon cancer, lung cancer, liver cancer, gastric cancer and melanoma.

10. A drug for preventing or treating tumors, characterized in that: The active ingredient of the drug includes the engineered cell vesicle nanovaccine for enhancing cancer immunotherapy according to claim 6 or 7.

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