A tumor whole cell biomimetic vesicle, and a preparation method and application thereof

Abstract

The present application relates to a kind of tumor whole cell biomimetic vesicle and its preparation method and application, belong to biological medicine technical field.The present application provides a kind of preparation method of tumor whole cell biomimetic vesicle, comprising the following steps: (1) tumor cell is mixed with BCG vaccine bacterial liquid, incubation, washing and obtains immunogenicity enhanced tumor cell;(2) the immunogenicity enhanced tumor cell is resuspended, high pressure extrusion, and purification obtains tumor whole cell biomimetic vesicle.The present application adopts live bacteria BCG to infect solid tumor cell, not only retains the whole antigen bank of tumor cell, but also can effectively trigger tumor cell to occur immunogenicity cell death, to generate signal molecule, and tumor whole antigen is delivered in cooperation, overcome the weak defect of single antigen immunogenicity, for breaking tumor immune tolerance, start strong specific anti-tumor immune response provides solid foundation, provides new train of thought for the research and development of solid tumor whole cell vaccine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a whole-cell biomimetic vesicle for tumors, its preparation method, and its application. Background Technology

[0002] Whole-cell tumor vaccines, containing a complete array of tumor antigens, eliminate the need for identifying dominant epitopes and significantly reduce the risk of immune escape due to tumor antigen heterogeneity, making them particularly suitable for tumors with high mutation burden, such as melanoma and bladder cancer. However, whole-cell vaccines often have weak immunogenicity and require the addition of exogenous adjuvants. Bacillus Calmette-Guérin (BCG), a live bacterial immunomodulator, is currently the gold standard for the clinical treatment of early-stage superficial bladder cancer. Studies have shown that specific stimuli (such as pathogen-associated molecular patterns, PAMPs) can induce long-term transcriptomic and epigenetic reprogramming of innate immune cells, generating "trained immunity (TI)." However, traditional direct local perfusion or injection of BCG results in significant systemic adverse reactions (such as BCG sepsis, severe local inflammation, and infection spread), greatly limiting its clinical application. Furthermore, achieving a deep and organic integration of BCG's potent adjuvant function with specific tumor whole-cell antigens remains a challenge in the fields of nanomedicine and vaccine delivery. Therefore, developing a nanoscale biomimetic vaccine that retains the full-cell antigen profile of tumors, possesses potent immune adjuvant function, and has high safety has significant clinical translational value and market prospects.

[0003] Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a whole-cell biomimetic vesicle for tumors, its preparation method and application, in order to solve the limitations of traditional BCG treatment methods in the prior art.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing whole-cell biomimetic vesicles for tumors, comprising the following steps: (1) Mix tumor cells with BCG bacterial solution to obtain a mixture, incubate for 12-48 h, and wash to obtain tumor cells with enhanced immunogenicity; (2) The tumor cells with enhanced immunogenicity were resuspended, squeezed under high pressure, and purified to obtain whole-cell biomimetic vesicles of tumor cells; The ratio of BCG to tumor cells in the mixture is 5-15:1.

[0006] Preferably, the OD of the BCG bacterial solution in step (1) is... 600 The value is 0.8 to 1.0.

[0007] Preferably, the tumor cells in step (1) include either bladder cancer cells or melanoma cells.

[0008] Preferably, the incubation temperature in step (1) is 35~39℃ and the percentage content of CO2 is 3~8%.

[0009] Preferably, the high-pressure extrusion method in step (2) is to use a polycarbonate membrane with a gradient pore size for high-pressure extrusion.

[0010] Preferably, the gradient apertures are 2~4μm, 0.5~0.7μm and 0.2~0.4μm from top to bottom.

[0011] This invention provides tumor whole-cell biomimetic vesicles prepared by the aforementioned preparation method.

[0012] This invention provides the use of the aforementioned tumor whole-cell biomimetic vesicles in the preparation of medicaments for the treatment or prevention of melanoma and / or bladder cancer.

[0013] Preferably, the drug includes a vaccine.

[0014] The present invention provides a vaccine for the prevention of melanoma and / or bladder cancer, the vaccine using phosphate buffer solution as a solvent and comprising components at the following concentrations; 50-600 μg / mL of tumor whole-cell biomimetic vesicles and 2.5-5 wt% trehalose; The tumor whole-cell biomimetic vesicle is the tumor whole-cell biomimetic vesicle.

[0015] The present invention has the following technical effects and advantages: This invention provides a whole-cell biomimetic vesicle for tumors, prepared by purifying BCG-infected tumor cells using high-pressure extrusion technology. This invention uses live BCG to infect solid tumor cells (such as melanoma cells and bladder cancer cells). Utilizing the strong immunostimulatory properties of BCG, it not only perfectly preserves the complete antigen library on the surface and inside of tumor cells but also effectively triggers immunogenic cell death (ICD) in tumor cells, thereby generating and enriching a large number of ICD-related danger signaling molecules. These signaling molecules, acting as potent natural adjuvants, synergistically deliver tumor whole antigens, overcoming the weakness of single antigen immunogenicity. This provides a solid foundation for breaking tumor immune tolerance and initiating a powerful specific anti-tumor immune response, offering a new approach for the development of whole-cell vaccines for solid tumors.

[0016] This invention reveals the mechanism of action and efficacy of the described biomimetic vesicles in vivo. The nanoscale physical size endows the biomimetic vesicles with extremely strong in vivo chemotaxis and targeting capabilities, enabling them to migrate efficiently to lymph nodes and bone marrow, significantly enhancing the uptake and presentation efficiency of antigen-presenting cells such as dendritic cells (DCs). Simultaneously, these biomimetic vesicles can activate a long-lasting, broad-spectrum training immune effect, promoting strong proliferation and maturation of T lymphocytes. Animal experiments show that applying these biomimetic vesicles to melanoma or bladder cancer models can effectively inhibit tumor growth and induce long-term immune memory, exhibiting remarkable anti-tumor efficacy. The biomimetic vesicles provided by this invention offer a safe and efficient new approach for the prevention and targeted treatment of solid tumors such as melanoma and bladder cancer. Attached Figure Description

[0017] Figure 1 Results of BCG uptake measurements in different tumor cells; Figure 2 The results are the physicochemical characterization of biomimetic vesicles; Figure 3 The results of the detection of BCG infection-induced immunogenic cell death in tumor cells; Figure 4 The results of measurements on the stimulation and activation of dendritic cells by biomimetic vesicle vaccines; Figure 5 Results of draining lymph nodes and isolated femoral / tibial imaging in different treatment groups; Figure 6 Tumor volume measurements in mice from different treatment groups; Figure 7 T cell subsets (CD4+) in the tumor tissue microenvironment of mice in different treatment groups + and CD8 + Flow cytometry results of the infiltration ratio; Figure 8 Systemic immune T cell subsets (CD4+) in the spleen of mice in different treatment groups + and CD8 + The proportion of flow cytometry detection results; Figure 9 The results of an assessment of the vaccine's ability to induce a "training immunity" effect in the bone marrow in vivo; Figure 10 This is the result of the in vivo safety evaluation of the biomimetic vesicle vaccine. Detailed Implementation

[0018] This invention provides a method for preparing whole-cell biomimetic vesicles for tumors, comprising the following steps: (1) Mix tumor cells with BCG bacterial solution to obtain a mixture, incubate for 12-48 h, and wash to obtain tumor cells with enhanced immunogenicity; The incubation time is preferably 30 hours; (2) The tumor cells with enhanced immunogenicity were resuspended, squeezed under high pressure, and purified to obtain whole-cell biomimetic vesicles of tumor cells; The ratio of BCG to tumor cells in the mixture is 5-15:1, preferably 8-12:1, and more preferably 10:1.

[0019] In this invention, the OD of the BCG bacterial solution described in step (1) 600 The value is 0.8~1.0, preferably 0.9.

[0020] In this invention, the tumor cells mentioned in step (1) include either bladder cancer cells or melanoma cells.

[0021] In this invention, the incubation temperature in step (1) is 35~39℃, and the percentage content of CO2 is 3~8%; The preferred temperature is 37°C, and the preferred percentage content of CO2 is 5%.

[0022] In this invention, the high-pressure extrusion method in step (2) is to use a polycarbonate membrane with gradient pore size for high-pressure extrusion.

[0023] In this invention, the gradient apertures are 2~4μm, 0.5~0.7μm and 0.2~0.4μm from top to bottom, and preferably 3μm, 0.6μm and 0.2μm from top to bottom.

[0024] This invention provides tumor whole-cell biomimetic vesicles prepared by the aforementioned preparation method.

[0025] This invention provides the use of the aforementioned tumor whole-cell biomimetic vesicles in the preparation of medicaments for the treatment or prevention of melanoma and / or bladder cancer.

[0026] In this invention, the drug includes a vaccine.

[0027] The present invention provides a vaccine for the prevention of melanoma and / or bladder cancer, the vaccine using phosphate buffer solution as a solvent and comprising components at the following concentrations; 50-600 μg / mL of tumor whole-cell biomimetic vesicles and 2.5-5 wt% trehalose; The preferred concentration of the tumor whole-cell biomimetic vesicles is 325 μg / mL; The preferred concentration of trehalose is 3.75 wt%. The tumor whole-cell biomimetic vesicle is the tumor whole-cell biomimetic vesicle.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] Preparation and characterization of BCG uptake by tumor cells and biomimetic vesicles (BMNVs and BBNVs)

[0031] (1) Verification of BCG uptake by tumor cells

[0032] Bladder cancer cells (MB49, purchased from Shanghai Jinyuan Biotechnology Co., Ltd., catalog number JY357) and melanoma cells (B16, purchased from Beina Chuanglian Biotechnology Co., Ltd., catalog number BNCC337628) were respectively divided into two groups at a density of 2.0 × 10⁻⁶. 5 Cells were inoculated at a density of 1 / mL and adherent to cell culture dishes. Fresh viable BCG bacteria (provided by the Public Health Clinical Center Affiliated to Fudan University) in the logarithmic growth phase (OD600=0.9) were added to cell culture dishes containing bladder cancer cells (MB49) and melanoma cells (B16) (the ratio of Wasabi-modified BCG bacteria to bladder cancer cells and melanoma cells was 10:1, i.e., the multiplicity of infection (MOI) was 10). The cells were incubated at 37℃ and 5% CO2 for 24 h. Cell uptake was observed using fluorescence microscopy at 3 h, 6 h, and 12 h, and parallel samples were collected at 0 h, 6 h, 12 h, and 24 h for flow cytometry analysis. Before analysis, cells were thoroughly washed with PBS to remove free BCG. The results of BCG uptake by different tumor cells are shown below. Figure 1 As shown, A represents fluorescence micrographs at 3h, 6h, and 12h, and B represents flow cytometry images at 0h, 6h, 12h, and 24h.

[0033] according to Figure 1 It can be seen that both MB49 and B16 cells can efficiently take up free BCG. Under fluorescence microscopy, a large amount of green fluorescence can be seen accumulating in the cytoplasm of the cells. Flow cytometry results show that the cell uptake positive rate is extremely high, proving that BCG has successfully entered the tumor cells.

[0034] (2) Preparation of biomimetic vesicles

[0035] MB49 and B16 cells were collected after the above incubation and resuspended in PBS buffer to obtain MB49 cell resuspensions and B16 cell resuspensions, respectively. The MB49 and B16 cell resuspensions were transferred to a pneumatic high-pressure extruder and repeatedly extruded three times sequentially through polycarbonate membranes with pore sizes of 3.0 μm, 0.6 μm, and 0.2 μm to obtain nanoscale MB49 and B16 tumor whole-cell biomimetic vesicles, respectively. To increase storage stability, the prepared biomimetic vesicles (adjusted to a final concentration of 500 μg / mL) were dispersed in PBS (pH 7.4), and trehalose (5 wt%) was added as a cryoprotectant. The vesicles were then aliquoted into 3 mL centrifuge tubes and stored at -80°C. The MB49-derived vaccine was designated BMNVs, and the B16-derived vaccine was designated BBNVs.

[0036] (3) Physicochemical characterization of biomimetic vesicles

[0037] 1 mL of each of the biomimetic vesicles (BBMVs) and BMNVs was placed in the sample cell of a laser particle size analyzer. The particle size, polydispersity index, and zeta potential of each sample were measured using dynamic light scattering (DLS). 100 μL of each of the BBMVs and BMNVs was added to 4% glutaraldehyde fixative and incubated overnight at 4°C to fix the biomimetic vesicle structure. The biomimetic vesicle suspension was then gently pipetted and removed, added to double-distilled water, and mixed thoroughly. The suspension was then dropped onto a carbon film copper grid using a dropper, and excess liquid was carefully blotted with filter paper. The copper grid was then observed under a transmission electron microscope at an accelerating voltage of 75 kV and a suitable magnification for imaging and recording. The physicochemical characterization results of the biomimetic vesicles are as follows: Figure 2 As shown in the figure, A represents the dynamic light scattering (DLS) particle size distribution of BMNVs, B represents the transmission electron microscope (TEM) image of BMNVs, C represents the dynamic light scattering (DLS) particle size distribution of BBNVs, and D represents the transmission electron microscope (TEM) image of BBNVs.

[0038] according to Figure 2 It can be seen that the particle size of both BBMVs and BMNVs is around 200nm, exhibiting a good normal distribution characteristic, and both show a regular spherical shape and have a complete membrane structure.

[0039] Example 2

[0040] (1) Detection of BCG infection-induced tumor cell immunogenic cell death (ICD)

[0041] Tumor cells (MB49 and B16) in logarithmic growth phase were sampled at a rate of 2 × 10⁻⁶.5 BCG cells were seeded at a density of [number] cells / well in 6-well culture plates pre-filled with sterile cell spreaders and incubated overnight at 37°C with 5% CO2. The following day, BCG culture was added to cell culture dishes containing bladder cancer cells (MB49) and melanoma cells (B16) (the ratio of BCG cells to bladder cancer cells and melanoma cells was 10:1, i.e., the multiple of infection (MOI) was 10), and incubated for 24 hours. An equal volume of complete culture medium without BCG was used as a blank control. After incubation, the culture medium was aspirated, and the cells were gently washed three times with pre-cooled PBS to remove free BCG. Subsequently, 4% paraformaldehyde (PFA) was added for fixation at room temperature for 15 minutes. After rinsing three times with PBS, blocking buffer containing 5% BSA was added and the cells were blocked at room temperature for 1 hour to block non-specific binding sites. The blocking solution was discarded, and anti-CRT primary antibody (Abcam, ab92516) diluted 1:400 with the blocking solution was added. The mixture was then incubated overnight at 4°C in a humidified chamber. The next day, the cells were washed three times with PBS (5 min each time), and HRP-labeled secondary antibody of the corresponding species (1:1000 dilution) was added. The cells were incubated at room temperature in the dark for 1 h. After washing, TSA (tyramine signal amplification) fluorescent substrate was added and incubated for 10 min in the dark for signal cascade amplification. Finally, DAPI staining solution was added and incubated at room temperature for 5 min to counterstain the cell nuclei. The slides were mounted with anti-fluorescence quenching mounting medium and observed and images were acquired under a laser confocal microscope (CLSM). The DAPI staining results for different treatment groups are shown below. Figure 3 As shown in A in the diagram.

[0042] according to Figure 3 As shown in Figure A, the CRT fluorescence signal of MB49 and B16 cells was weak at 0h; with the extension of BCG treatment time, the CRT-positive fluorescence signal on the cell surface gradually increased at 12h and 24h, becoming more pronounced at 24h. Tumor cells treated with BCG for 24h exhibited dense, punctate clusters of strong CRT-positive fluorescence signals on their cell membrane surface. This definitively demonstrates that intracellular infection with BCG effectively triggered immunogenic cell death (ICD) in tumor cells, causing a large amount of the danger signal molecule CRT to extravasate to the cell membrane surface, providing a strong "Eat me" signal for subsequent antigen uptake.

[0043] (2) Flow cytometry detection of expression levels of major histocompatibility complex-I (MHC-I)

[0044] MB49 and B16 tumor cells were seeded into 6-well plates and incubated with live BCG at an MOI of 10 for 24 h. An equal volume of complete culture medium without BCG was used as a blank control. After incubation, cells were collected using a mild, EDTA-free digestion buffer to maximize the protection of cell membrane antigens. The collected cell suspension was centrifuged at 300 g for 5 min at 4 °C, the supernatant was discarded, and the cells were resuspended in pre-chilled flow cytometry buffer (FACS buffer) containing 2% FBS and washed twice. The cell concentration was adjusted to 1 × 10⁻⁶ cells / well. 6 For each cell / tube, Fc receptor blocker (anti-CD16 / 32) was first added and incubated on ice for 10 min to reduce non-specific antibody binding. Then, an appropriate amount of fluorescently labeled antibody PE anti-mouse H-2Kb / H-2Db (Biolegend, 114607, specifically targeting MHC class I molecules) was added, and the cells were incubated at 4°C in strict light-protected conditions for 30 min. After incubation, 1 mL of pre-chilled FACS buffer was added, and the cells were washed twice by centrifugation to thoroughly remove free antibody. Finally, the cell pellet was resuspended in 300 μL of PBS and placed in a flow cytometer for data acquisition. For data analysis, viable cell populations were identified using forward / side-scattered light (FSC / SSC), and binucleated cells were excluded. The fluorescence signal from the PE channel was extracted for mean fluorescence intensity (MFI) analysis. The mean fluorescence intensity results for different treatment groups are shown below. Figure 3 As shown in B in the diagram.

[0045] according to Figure 3 As shown in Figure B, with the BCG treatment time extended from 0h to 6h, 12h, and 24h, the fluorescence peaks of MHC-I molecules on the surface of MB49 and B16 cells generally shifted to the right. This indicates that because the vesicle preparation process completely preserves the intact membrane structure, the significant increase in the expression level of MHC-I on the cell surface is inherited by the vesicles, thereby greatly enhancing the target basis and antigen presentation potential of infected tumor cells (and subsequently prepared vesicle vaccines) to be recognized by the body's immune system.

[0046] Example 3

[0047] Biomimetic vesicle vaccines stimulate and activate dendritic cells (BMDCs).

[0048] (1) Detection of BMDCs' ability to take up vaccines

[0049] Mice bone marrow cells were extracted and induced to form immature bone marrow-derived dendritic cells (BMDCs). To investigate the ability of BMDCs to take up biomimetic vesicle vaccines (BMNVs and BBNVs), the lipid-soluble fluorescent dye DiO (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number C1038) was used as a fluorescent probe. Following the kit instructions, MNVs (using PBS as solvent, including MB49 cells at a final concentration of 500 μg / mL and trehalose at a final concentration of 5 wt%), BMNVs, BNVs (using PBS as solvent, including B16 cells at a final concentration of 500 μg / mL and trehalose at a final concentration of 5 wt%), and BBNVs were stained in the dark. After labeling, the cells were washed with PBS and centrifuged three times to remove excess free DiO, yielding labeled vaccines with green fluorescence.

[0050] The DiO-labeled MNV, BMNV, BNV, and BBNV were added to confocal culture dishes inoculated with BMDCs and incubated for 12 hours. After incubation, the supernatant was removed, and 1 mL of pre-warmed Lyso-Tracker Red working solution (37°C) was added. The cells were incubated at room temperature in the dark for at least 40 minutes to stain the lysosomes of the BMDCs with red fluorescence. The working solution was discarded, and the cells were washed three times with PBS. Pre-cooled 4% paraformaldehyde fixative was added to fix the cells for 30 minutes, followed by three washes with PBS. Finally, DAPI staining solution was added and the cells were incubated at 37°C in the dark for 5 minutes to stain the nuclei of the BMDCs with blue fluorescence. The DAPI was discarded, and the cells were washed three times with PBS for 5 minutes each time. After treatment, the uptake of the biomimetic vesicle vaccine by the BMDCs was observed under a laser confocal microscope (CLSM). The results are as follows: Figure 4 As shown in A in the diagram.

[0051] according to Figure 4 As shown in A, dense vaccine fluorescence signals were observed in the cytoplasm of BMDCs, proving that these biomimetic vesicles (BBNV and BMNV) are easily and efficiently taken up by antigen-presenting cells.

[0052] (2) Maturity detection of BMDCs

[0053] To evaluate the activation ability of biomimetic vesicle vaccines on BMDCs, we measured the expression of co-stimulatory molecules CD80 and CD86 on the surface of BMDCs. BMDCs were processed at a ratio of 2 × 10⁻⁶. 6Cells were seeded into 6-well plates at a density of cells / well. The following day, different groups of vaccines were added to each well: for MB49-derived vesicles, a Control group (with PBS as a control), a MNV (50 μg / mL) group, and BMNV groups at concentrations of 12.5 μg / mL, 25 μg / mL, and 50 μg / mL were set up; for B16-derived vesicles, a Control group (PBS), a BNV (50 μg / mL) group, and BBNV groups at concentrations of 12.5 μg / mL, 25 μg / mL, and 50 μg / mL were set up. After 48 hours of incubation, the centrifuged cell pellet was resuspended in 100 μL of PBS, and 1 μL of CD11c, CD80, and CD86 flow cytometry antibodies were added. After thorough mixing, the cells were incubated on ice in the dark for 30 minutes. After incubation, the cells were resuspended in PBS and centrifuged twice to ensure the removal of excess antibodies. Finally, the cells were resuspended in 500 μL of pre-chilled PBS and transferred to flow cytometry sample tubes. The expression of CD11c, CD80, and CD86 markers on the surface of mature bone marrow-derived dendritic cells (BMDCs) was detected by flow cytometry (FCM) to assess the activation level of these vaccines on DCs. Results are as follows: Figure 4 As shown in B in the diagram.

[0054] according to Figure 4 As shown in B, compared with the Control group and the corresponding MNV or BNV group, the proportion of CD80 and CD86 double positive cells on the surface of BMDCs was generally increased after BMNV and BBNV treatment, and the proportion increased with the increase of vesicle concentration from 12.5 μg / mL to 25 μg / mL and 50 μg / mL, indicating that biomimetic vesicles can promote the maturation of BMDCs.

[0055] (3) In vivo targeting (lymph node and bone marrow) verification of biomimetic vesicle vaccines

[0056] After staining the biomimetic vesicle vaccine with DiR, it was subcutaneously injected into the left back of healthy C57BL / 6 mice. Mice were anesthetized with isoflurane and then randomly divided into three groups: a blank control group (PBS), a control group (MNV and BNV), and a vaccine treatment group (BMNV and BBNV), with three mice in each group. The distribution of red fluorescence in the lymph node region of the mice was observed using an in vivo imaging system. Twenty-four hours after administration, the mice were euthanized, and the draining lymph nodes and femur / tibia were dissected for in vitro imaging. The results of in vitro imaging of the draining lymph nodes and femur / tibia in different treatment groups are shown below. Figure 5 As shown in the figure, A represents the in vitro imaging results of draining lymph nodes in different treatment groups, and B represents the in vitro imaging results of femur / tibia in different treatment groups.

[0057] according to Figure 5It can be seen that the vaccine can migrate effectively after injection. In vivo imaging showed strong fluorescence enrichment in the draining lymph node area; in vitro bone marrow imaging also detected obvious fluorescence signals, proving that the biomimetic vaccine not only has excellent lymph node targeting ability, but can also penetrate the physiological barrier and enrich deep into the bone marrow microenvironment.

[0058] Example 4

[0059] In vivo antitumor efficacy and immune microenvironment remodeling of biomimetic vesicle vaccines

[0060] (1) Establishment of mouse tumor treatment model and evaluation of efficacy

[0061] Healthy 6-week-old C57BL / 6 mice were randomly divided into three groups of 6 mice each: a blank control group (PBS), a control group (MNV and BNV), and a vaccine treatment group (BMNV and BBNV). The vaccine was administered via subcutaneous injection at multiple sites, with three injections given 7 days apart, at a dose of 50 μg per mouse. Seven days after the final immunization, mice in each group were subcutaneously inoculated with the same number of viable bladder cancer MB49 cells and melanoma B16 cells on the right back to induce tumor challenge. Tumor volume measurements after inoculation in the different treatment groups are shown below. Figure 6 As shown in the figure, A represents the tumor volume measurement results of bladder cancer MB49 in different treatment groups, and B represents the tumor volume measurement results of melanoma B16 in different treatment groups.

[0062] according to Figure 6 It was found that in the control group mice, the tumors grew exponentially after inoculation with tumor cells; while in mice that had been immunized with BMNVs and BBNVs beforehand, a strong specific immune defense had been established in their bodies, tumor growth was significantly inhibited, the onset of tumors was greatly delayed, and many mice did not develop tumors at all. This experiment confirms that the biomimetic vesicle vaccine of this invention has excellent anti-tumor preventive effects.

[0063] (2) Detection of tumor microenvironment and systemic immune T cell subsets

[0064] At the end of the experiment, the mice used in the prevention model were sacrificed, and their tumor tissue and spleen were aseptically isolated and prepared into a single-cell suspension through grinding and digestion. CD3 and CD4 were then added. + and CD8 + Flow cytometry antibody staining was used to detect T cell infiltration and activation.

[0065] Preparation of spleen single-cell suspension: A whole spleen was placed in a sterile Petri dish, and pre-chilled PBS buffer was added. The spleen tissue was gently ground on a 70 μm cell sieve using a syringe core. The filtrate was collected and centrifuged at 300 g for 5 min at 4 °C, and the supernatant was discarded. An appropriate amount of erythrocyte lysis buffer was added to the pellet, and lysis was performed at room temperature for 5 min to remove erythrocytes. Then, excess PBS was quickly added to terminate the lysis. After washing again by centrifugation, the spleen cells were resuspended in flow cytometry staining buffer (FACS Buffer) containing 2% fetal bovine serum (FBS) for later use.

[0066] Preparation of single-cell suspension of tumor microenvironment: Tumor tissues from mice in each group were collected, the surface capsule and necrotic tissue were peeled off, and the tissues were cut into 2mm pieces using ophthalmic scissors. 3 Tissue fragments were transferred to centrifuge tubes and serum-free DMEM digestion buffer containing collagenase IV (1 mg / mL) and deoxyribonuclease I (0.1 mg / mL) was added. The tubes were incubated at 37°C in a shaker at 200 rpm for 60 min. After digestion, complete culture medium containing 10% FBS was added to terminate the digestion. The digestion buffer was filtered through a 70 μm cell filter, and the cell pellet was collected by centrifugation. After washing twice with PBS, the pellet was resuspended in FACS Buffer for later use.

[0067] Flow cytometry antibody staining and detection: Add the prepared spleen and tumor single-cell suspensions (adjusting the cell concentration to 1×10⁻⁶) to the solution. 6 First, anti-mouse CD16 / 32 antibody (Fc Block) was added to each tube, and the cells were incubated at 4°C in the dark for 15 min to block the Fc receptors on the cell surface, reducing the non-specific binding of subsequent fluorescent antibodies. After centrifugation and washing to remove the blocking solution, a combination of specific surface marker fluorescent antibodies was added, including anti-mouse CD3 fluorescent antibody, anti-mouse CD4 fluorescent antibody, and anti-mouse CD8 fluorescent antibody. After thorough mixing, the cells were incubated at 4°C or on ice in the dark for 30 min. After incubation, 1 mL of pre-chilled PBS buffer was added, and the cells were centrifuged and washed twice to completely remove unbound free antibodies. Finally, the cells were resuspended in 300 μL of PBS, and data were collected using flow cytometry. The T cell subsets (CD4+, CD ... + and CD8 + The flow cytometry results of the infiltration ratio are as follows: Figure 7 As shown in the figure, A represents the results of BMNVs treatment, and B represents the results of BBNVs treatment; systemic immune T cell subsets (CD4+) in the spleen of mice in different treatment groups. + and CD8 + The flow cytometry results of the proportion are as follows: Figure 8As shown in the figure, A represents the treatment result of BMNVs, and B represents the treatment result of BBNVs.

[0068] according to Figure 7 and Figure 8 It can be seen that, in the tumor microenvironment, both BMNVs and BBNVs prevention groups can significantly increase CD4 levels. + and CD8 + The increased infiltration of effector T cells successfully reversed the immunosuppressive state at the tumor site. Simultaneously, in the spleen, CD4, representing systemic immunity, was significantly increased. + and CD8 + The proportion of T cells also increased significantly, proving that early immunization with the vaccine successfully evoked strong and long-lasting systemic specific anti-tumor immune memory in mice.

[0069] Example 5

[0070] Bionic vesicle vaccines induce a bone marrow "training immunity" effect in vivo.

[0071] To systematically evaluate the vaccine's ability to induce innate immune reprogramming (i.e., "trained immunity") in the bone marrow, single cells were extracted from mouse bone marrow, and precise flow cytometry analysis was performed to determine the differentiation trajectories of hematopoietic stem / progenitor cells within the bone marrow. The expansion of granulocyte-macrophage progenitors (GMPs) was examined. At the end of Example 4, mice in each group were sacrificed, and under aseptic conditions, the hind limb muscles were dissected, and both tibias and femurs were completely removed. The ends of the bones were cut to expose the medullary cavity. Using a syringe equipped with a 25G needle, pre-cooled PBS buffer containing 2% fetal bovine serum (FBS) was drawn and repeatedly flushed the medullary cavity until the bone shaft turned pale. The flushed bone marrow cell clusters were collected, gently dispersed by pipetting, and then the cell suspension was passed through a 70μm cell sieve to remove bone debris and tissue clots. Centrifuge at 300g for 5 min at 4°C, discard the supernatant; add an appropriate amount of erythrocyte lysis buffer (ACK Lysis Buffer) and lyse on ice for 3 min, then add excess PBS to terminate lysis. After centrifugation and washing, resuspend the bone marrow cells in flow cytometry staining buffer (FACS Buffer) and adjust the cell concentration to approximately 1×10⁻⁶ cells / mL. 7 per mL.

[0072] Take 100 μL of the cell suspension prepared above and add it directly to the multicolor flow cytometry antibody combination for staining.

[0073] Lineage (Lin) negative antibody mixture: FITC anti-mouse Lineage Cocktail (Biolegend, 133301) was added to exclude all mature peripheral blood lineage hematopoietic cells. Simultaneously, APC anti-mouse CD117 (c-kit) (Biolegend, 135108), APC / Cyanine7 anti-mouse Ly-6A / E (Sca-1) (Biolegend, 108125), PE anti-mouse CD34 (Biolegend, 128609), and PerCP / Cyanine5.5 anti-mouse CD16 / 32 (Biolegend, 101323) were added and mixed thoroughly. The mixture was then incubated at 4°C on ice in the dark for 45 min. After incubation, the cells were washed twice with 1 mL of FACS Buffer, resuspended, and data were acquired using flow cytometry.

[0074] Gate analysis was performed strictly according to the following hierarchical logic: First, the viable cell population was delineated using forward angle scattering (FSC-A) and side angle scattering (SSC-A), and then doublets were excluded using FSC-W / FSC-H. Within the viable cell population, Lineage-negative cells (Lin...) were delineated. - (A population of) excluding interference from mature cells. In Lin - Inside the gate, construct a scatter plot of c-Kit (APC channel) corresponding to Sca-1 (APC / Cyanine7 channel) to delineate Lin. - c-Kit + Sca-1 - The LSK cell population (referred to as LSK cells) represents myeloid progenitor cells. Finally, within the LSK cell phylum, a scatter plot was constructed showing CD34 (PE channel) corresponding to CD16 / 32 (PerCP / Cyanine 5.5 channel). CD34... + CD16 / 32 + The double-positive cell population was precisely defined as granulocyte-monocyte progenitor cells (GMPs). (That is, the complete phenotype of GMPs cells was rigorously confirmed as Lin.) - c-Kit + Sca-1 - CD16 / 32 + CD34 + ).

[0075] The evaluation results of the vaccine's induction of the bone marrow's "training immunity" effect in vivo are as follows: Figure 9 As shown in the figure, A represents the BMNVs group and B represents the BBNVs group.

[0076] according to Figure 9 It was found that mice vaccinated with BMNVs and BBNVs showed a significantly increased proportion of GMPs cells with the aforementioned strict phenotypic definition in their bone marrow microenvironment compared to the PBS control group. This result definitively confirms at the cellular differentiation level that the biomimetic vesicle vaccine prepared in this invention not only generates antigen-specific immunity in the periphery but also successfully flows back into the bone marrow, reshaping the differentiation trajectory of bone marrow hematopoietic stem / progenitor cells and inducing a strong and durable broad-spectrum "training immunity" defense effect from the source of innate immunity.

[0077] Example 6

[0078] In vivo safety evaluation of biomimetic vesicle vaccines

[0079] Direct use of live bacterial BCG often carries a very high risk of local ulceration and systemic sepsis. To verify the absolute safety of the nanoscale live-free vesicle vaccine prepared by high-pressure extrusion technology in vivo, rigorous histopathological testing (H&E staining) was performed. Specifically, at the end of the experiment in Example 4, mice in each group were euthanized. The main metabolic and immune organs, such as the heart, liver, spleen, lungs, and kidneys, of the mice were quickly dissected and completely removed. After rinsing the surface of the organs with pre-cooled PBS to remove blood, they were immediately immersed in at least 10 times their volume of 4% paraformaldehyde (PFA) fixative and fixed at room temperature for 48 hours to preserve cell structure and tissue morphology. The fixed tissue blocks were rinsed with running water and then sequentially dehydrated with graded ethanol using a fully automated tissue dehydrator. After dehydration, xylene was used for clearing until the tissue was translucent. Finally, the tissue was immersed in molten liquid paraffin (60°C, 2 hours) and solidified into paraffin tissue blocks using an embedding machine. Paraffin blocks were cut into continuous thin slices with a thickness of 5 μm, attached to anti-detachment glass slides, dewaxed, rehydrated, and stained with H&E. After dehydration with graded ethanol and clearing with xylene, neutral resin was added and the slides were mounted. Pathological observation was performed using a high-resolution optical microscope, and in vivo safety was evaluated. The in vivo safety evaluation results of the biomimetic vesicle vaccine are as follows: Figure 10 As shown in the figure, A represents the BMNVs treatment group and B represents the BBNVs treatment group.

[0080] according to Figure 10It was found that mice immunized multiple times with BMNVs and BBNVs maintained normal homeostasis in the structures of their major organs and tissues. Myocardial fibers were tightly and neatly arranged, liver lobules were intact without ballooning degeneration, the white and red pulp of the spleen were clearly demarcated, alveolar walls showed no thickening or inflammatory exudate, and glomeruli and tubules exhibited regular morphology. No signs of cell degeneration, focal necrosis, congestion, edema, or abnormal inflammatory cell infiltration were observed in any of the sections. These results, at the histological level, fully demonstrate the extremely high biocompatibility and excellent drug development potential of this nanovesicle vaccine.

[0081] As can be seen from the above embodiments, the present invention provides a tumor whole-cell biomimetic vesicle, its preparation method, and its application. The present invention uses live bacterial BCG to infect solid tumor cells, which not only preserves the complete antigen library of tumor cells but also effectively triggers immunogenic cell death in tumor cells, thereby generating signaling molecules that are synergistically delivered with the tumor whole antigen. This overcomes the weakness of single antigen immunogenicity, providing a solid foundation for breaking tumor immune tolerance and initiating a strong specific anti-tumor immune response, and offering a new approach for the development of whole-cell vaccines for solid tumors.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing whole-cell biomimetic vesicles for tumors, characterized in that, Includes the following steps: (1) Mix tumor cells with BCG bacterial solution to obtain a mixture, incubate for 12-48 h, and wash to obtain tumor cells with enhanced immunogenicity; (2) The tumor cells with enhanced immunogenicity were resuspended, squeezed under high pressure, and purified to obtain whole-cell biomimetic vesicles of tumor cells; The ratio of BCG to tumor cells in the mixture is 5-15:

1.

2. The preparation method according to claim 1, characterized in that, The OD of the BCG bacterial solution described in step (1) 600 The value is 0.8 to 1.

0.

3. The preparation method according to claim 1, characterized in that, The tumor cells mentioned in step (1) include one of the bladder cancer cells and melanoma cells.

4. The preparation method according to claim 1, characterized in that, The incubation temperature in step (1) is 35~39℃, and the percentage content of CO2 is 3~8%.

5. The preparation method according to claim 1, characterized in that, The high-pressure extrusion method described in step (2) is to use a polycarbonate membrane with a gradient pore size for high-pressure extrusion.

6. The preparation method according to claim 5, characterized in that, The gradient apertures are 2~4μm, 0.5~0.7μm and 0.2~0.4μm from top to bottom.

7. Tumor whole-cell biomimetic vesicles prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the tumor whole-cell biomimetic vesicles according to claim 7 in the preparation of medicaments for the treatment or prevention of melanoma and / or bladder cancer.

9. The application according to claim 8, characterized in that, The drugs mentioned include vaccines.

10. A vaccine for the prevention of melanoma and / or bladder cancer, characterized in that, The vaccine uses phosphate buffer solution as a solvent and includes components at the following concentrations; 50-600 μg / mL of tumor whole-cell biomimetic vesicles and 2.5-5 wt% trehalose; The tumor whole-cell biomimetic vesicle is the tumor whole-cell biomimetic vesicle as described in claim 7.

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