Nanomotor driven tumor antigen captured in-situ vaccine as well as construction method and application of nanomotor driven tumor antigen captured in-situ vaccine

Abstract

The invention discloses an in-situ vaccine for tumor antigen capture driven by a nano motor as well as a construction method and application of the in-situ vaccine. The preparation method comprises the following steps: mixing prepared enzyme-immobilized dendritic silicon dioxide nanoparticles and drug-loaded bacterial outer membrane vesicles, extruding, carrying out solid-liquid separation, dispersing the obtained solid with a solvent, co-incubating the obtained suspension with pH-responsive membrane disrupting peptides and receptor molecules of targeted tumor cells, and carrying out freeze-drying to obtain the drug-loaded dendritic silicon dioxide nano-particles with the pH-responsive membrane disrupting peptides. The in-situ vaccine captured by the tumor antigen driven by the nano motor is obtained. The preparation method is green, simple and easy to operate. The in-situ vaccine can rapidly capture, enrich and release tumor-associated antigens and deliver the tumor-associated antigens to APCs, so that the reduction of curative effect caused by tumor heterogeneity and antigen degradation is avoided, the antigen presentation efficiency is remarkably improved, and the immune response is further enhanced; and the method is suitable for various tumors and has universality.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an in situ vaccine for capturing tumor antigens driven by nanomotor, and a construction method and application thereof. Background Art

[0002] Cancer vaccines, typically composed of antigens and adjuvants, have shown considerable promise in eliminating tumors, establishing long-lasting anti-tumor memory, and avoiding nonspecific or adverse reactions. Traditional cancer vaccines use purified or prepared antigens and classic adjuvants (aluminum hydroxide, emulsions, liposomes, cytokines, toll-like receptor agonists, saponins), but this strategy has proven complex and expensive, and also faces challenges such as low antigen immunogenicity and tumor heterogeneity, resulting in limited anti-tumor immune efficacy, incomplete tumor treatment, and a high risk of recurrence and metastasis.

[0003] In contrast, in situ cancer vaccines attack tumor cells, convert them into tumor-associated antigens, activate antigen-presenting cells (APCs) and T lymphocytes, and further identify and eliminate tumor cells. This approach does not require the identification and separation of patient-specific tumor-associated antigens, solves the challenges posed by tumor heterogeneity, and provides a wider range of therapeutic application possibilities. However, the efficacy of in situ vaccines is still limited. Although inducing immunogenic cell death (ICD) of tumor cells can release a large amount of tumor-associated antigens, due to the body's innate immune clearance and the tumor immunosuppressive microenvironment, tumor-associated antigens are rapidly degraded and cleared, resulting in dendritic cells (DCs) being unable to fully absorb, process and present antigens, thereby hindering the anti-tumor immune response. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for constructing an in situ vaccine that captures tumor antigens driven by nanomotors.

[0005] Another object of the present invention is to provide an in situ vaccine for tumor antigen capture driven by the nanomotor obtained by the above construction method.

[0006] Another object of the present invention is to provide an application of the above nanomotor-driven in situ vaccine for tumor antigen capture.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for constructing an in situ vaccine for nanomotor-driven tumor antigen capture comprises the following steps:

[0009] (1) Preparation of enzyme-immobilized dendritic silica nanoparticles:

[0010] A. mixing cetyltrimethylammonium chloride (CTAC) solution, triethylamine (TEA) solution and water and stirring to obtain a mixed solution;

[0011] B. adding tetraethyl orthosilicate (TEOS) and cyclohexane to the bottom of the aqueous phase of the mixed solution and stirring, followed by solid-liquid separation, washing the obtained solid, and then removing the template hexadecyltrimethylammonium chloride to obtain porous nanoparticles;

[0012] C. functionalizing the porous nanoparticles with triethoxysilane (APTES) to obtain amino-functionalized nanoparticles;

[0013] D. dropping the enzyme solution into the amino-functionalized nanoparticle solution with stirring, separating the solid and the liquid, and washing the obtained solid to obtain enzyme-immobilized dendritic silica nanoparticles;

[0014] (2) Preparation of drug-loaded bacterial outer membrane vesicles: The bacterial culture supernatant is centrifuged by ultracentrifugation to obtain bacterial outer membrane vesicles; the drug solution is then mixed with the bacterial outer membrane vesicle solution and extruded through a liposome extruder to remove unloaded drugs, thereby obtaining drug-loaded bacterial outer membrane vesicles;

[0015] (3) Preparation of in situ vaccines driven by nanomotor-driven tumor antigen capture:

[0016] A. mixing a dispersion of enzyme-immobilized dendritic silica nanoparticles and drug-loaded bacterial outer membrane vesicles, extruding, and performing solid-liquid separation. The obtained solid is dispersed in a solvent to obtain a suspension.

[0017] B. The suspension is co-incubated with a pH-responsive membrane-disrupting peptide and a receptor molecule targeting tumor cells to obtain an in situ vaccine that captures tumor antigens driven by nanomotor.

[0018] The concentration of the cetyltrimethylammonium chloride solution in step (1)A is preferably 20-30% wt; more preferably 25% wt.

[0019] The concentration of the triethylamine solution described in step (1) A is preferably 3-4% wt; more preferably 3.5% wt.

[0020] The water described in step (1)A is preferably deionized water or ultrapure water.

[0021] The stirring conditions in step (1)A are preferably stirring at 50-70°C and 300-500 rpm for 30-90 min; more preferably stirring at 60°C and 400 rpm for 60 min.

[0022] The stirring conditions in step (1) B are preferably stirring at 50-70° C. and 300-500 rpm for 10-15 h; more preferably stirring at 60° C. and 400 rpm for 12 h.

[0023] The solid-liquid separation method in step (1)B is preferably centrifugation.

[0024] The centrifugal conditions are preferably 8000-12000 rpm for 20-40 min; more preferably 10000 rpm for 30 min.

[0025] The washing solvent in step (1)B is preferably ethanol.

[0026] The removal method described in step (1)B is preferably calcination.

[0027] The calcination conditions are preferably calcined at 500-600°C for 5-7 hours; more preferably calcined at 550°C for 6 hours.

[0028] The specific steps of functionalizing the porous nanoparticles with triethoxysilane in step (1) C are preferably as follows: dispersing the porous nanoparticles in ethanol, adding triethoxysilane, stirring evenly, and then refluxing, and separating the solid and liquid to obtain solid amino-functionalized nanoparticles.

[0029] The amount of ethanol used is preferably such that the volume (mL) of ethanol is 140 to 150 times the mass (g) of the porous nanoparticles.

[0030] The reflux condition is preferably reflux at 70-90° C. for 8-16 hours; more preferably reflux at 80° C. for 10-12 hours.

[0031] The solid-liquid separation method is preferably centrifugation.

[0032] The enzyme in step (1)D is an enzyme that degrades DNA or an enzyme that degrades ATP.

[0033] The enzyme that degrades DNA is preferably DNA enzyme (DNase); more preferably DNase I.

[0034] The enzyme that degrades ATP is preferably ATPase.

[0035] The concentration of the enzyme solution in step (1) D is preferably 4-6 mg / mL; more preferably 5 mg / mL.

[0036] The concentration of the amino-functionalized nanoparticle solution in step (1)D is preferably 8-12 mg / mL; more preferably 10 mg / mL.

[0037] The stirring condition in step (1) D is preferably stirring at 2-8° C. for 10-16 h; more preferably stirring at 4° C. for 12 h.

[0038] The solid-liquid separation method is preferably centrifugation.

[0039] The centrifugal conditions are preferably 6000-10000 rpm for 5-15 min; more preferably 8000 rpm for 10 min.

[0040] The washing solvent in step (1)D is preferably PBS.

[0041] In step (1):

[0042] The hexadecyltrimethylammonium chloride, triethylamine, ethyl silicate and cyclohexane are preferably mixed in a ratio of 1 g: 0.03-0.04 g: 1.0-1.5 mL: 8.5-9.0 mL; more preferably, in a ratio of 1 g: 0.035 g: 1.25 mL: 8.75 mL.

[0043] The amount of triethoxysilane used is preferably calculated based on 3.0 to 3.5 mL of triethoxysilane per gram of porous nanoparticles.

[0044] The amount of the enzyme is preferably calculated based on a mass ratio of 5:250 to 300 between the enzyme and the amino-functionalized nanoparticles; more preferably, the amount of the enzyme is calculated based on a mass ratio of 5:270 to 280 between the enzyme and the amino-functionalized nanoparticles.

[0045] The ultracentrifugation conditions in step (2) are preferably as follows: centrifugation at 120,000 to 180,000 g for 1 to 3 h at 2 to 8° C.; more preferably, centrifugation at 150,000 g for 2 h at 4° C.

[0046] The bacteria described in step (2) is preferably at least one of Salmonella enterica, lactic acid bacteria, attenuated Salmonella typhimurium, Shigella, Escherichia coli and Bifidobacterium; more preferably attenuated Salmonella.

[0047] The bacterial supernatant in step (2) is preferably prepared by the following steps: culturing the bacteria to the logarithmic growth phase to obtain a culture solution; and concentrating the liquid obtained by removing the bacterial cells from the culture solution.

[0048] The specific preferred method of removing the bacteria is as follows: first centrifugation, then filtration.

[0049] The centrifugation condition is preferably 3000-5000×g for 5-15 min; more preferably 4000×g for 10 min.

[0050] The drug described in step (2) is an antitumor drug; preferably a chemical drug; more preferably mitoxantrone (MTO) or mitoxantrone hydrochloride.

[0051] The concentration of the drug solution in step (2) is preferably 80-120 μg / mL; more preferably 100 μg / mL.

[0052] The concentration of the bacterial outer membrane vesicle solution in step (2) is preferably 20 to 30 μg / mL; more preferably 25 μg / mL.

[0053] The drug described in step (2) and the bacterial outer membrane vesicles are preferably mixed at a mass ratio of 1.5 to 2.5:1; more preferably at a mass ratio of 2:1.

[0054] The mixing conditions in step (2) are preferably stirring at 35-40° C. for 3-5 h; more preferably stirring at 37° C. for 4 h.

[0055] The extrusion described in step (2) used a 220 nm polycarbonate film.

[0056] The number of extrusions in step (2) is preferably 6 to 8 times; more preferably 7 times.

[0057] The removal of unloaded drugs in step (2) is achieved by ultrafiltration using a 100 kDa ultrafiltration membrane.

[0058] The concentration of the enzyme-immobilized dendritic silica nanoparticle dispersion in step (3)A is preferably 0.5 to 1.5 mg / mL; more preferably 1 mg / mL.

[0059] The concentration of the drug-loaded bacterial outer membrane vesicles described in step (3) A is preferably 0.4-0.6 mg / mL; more preferably 0.5 mg / mL.

[0060] The mixing method described in step (3)A is preferably vortex mixing.

[0061] The extrusion described in step (3)A used a 220 nm polycarbonate film.

[0062] The number of extrusions in step (3)A is preferably 6 to 8 times; more preferably 7 times.

[0063] The solid-liquid separation method in step (3)A is preferably centrifugation.

[0064] The centrifugal conditions are preferably 6000-10000 g for 10-30 min; more preferably 8000 g for 20 min.

[0065] The solvent described in step (3)A is preferably PBS; more preferably PBS at 2-8°C.

[0066] The amino acid sequence of the pH-responsive membrane-disrupting peptide described in step (3)B is as follows: FLEHLIPPHVIHGLVHAIHH-NH2.

[0067] The receptor molecule targeting tumor cells in step (3)B is preferably DSPE-PEG 2000 -FA.

[0068] The enzyme-immobilized dendritic silica nanoparticles, drug-loaded bacterial outer membrane vesicles, pH-responsive membrane-destroying peptides and tumor cell-targeting receptor molecules described in step (3) are preferably mixed in a mass ratio of 1-3:1:05-1.0:0.8-1.2; preferably in a mass ratio of 2:1.0:0.8:1.0.

[0069] The incubation time in step (3) B is preferably 10 to 16 hours, more preferably 12 hours.

[0070] An in situ vaccine for tumor antigen capture driven by a nanomotor is obtained by the above-mentioned construction method.

[0071] The use of the nanomotor driven tumor antigen capture in situ vaccine in the preparation of anti-tumor drugs; preferably comprising the following steps: injecting the nanomotor driven tumor antigen capture in situ vaccine into the tumor; wherein the surface folic acid (i.e. DSPE-PEG 2000 -FA in the FA can target tumor cells and achieve effective delivery of the capture system. The pH-responsive membrane-disrupting peptide on the surface undergoes conformational changes in the acidic microenvironment of the tumor, destroying the bacterial outer membrane vesicles and releasing anti-tumor drugs (such as mitoxantrone), inducing the immunogenic death of tumor cells and promoting the in situ generation of tumor-associated antigens. The enzyme-immobilized dendritic silica nanoparticles tend to move in the direction of enriching immunogenic dead cells and capture tumor-associated antigens, thereby improving antigen utilization, achieving co-delivery of adjuvants and antigens, further activating T cells, and improving the efficacy of cancer immunotherapy.

[0072] The present invention has the following advantages and effects compared to the prior art:

[0073] (1) As a new type of nanocarrier, nanomotors, with their active motion ability, can convert energy into mechanical motion, increase the probability of contact with released antigens, and can quickly capture and enrich released tumor-associated antigens and deliver them to APCs, thereby significantly improving the efficiency of antigen presentation and further enhancing the immune response. The in situ vaccine based on nanomotor-driven tumor antigen capture constructed by the present invention utilizes the motion ability of nanomotors to capture the patient's own antigens, avoiding the reduction in efficacy caused by tumor heterogeneity and antigen degradation; secondly, the preparation method is green, simple, and easy to operate; and the in situ vaccine of the present invention is applicable to a variety of tumors and has universal applicability.

[0074] (2) The unique structural design of the nanomotor gives it a large specific surface area and adjustable loading capacity, which can efficiently load captured tumor-associated antigens and adjuvants, further enhancing the immune activation ability.

[0075] (3) The biocompatibility of nanomotors ensures the safety of in vivo applications. Through these characteristics, the application of nanomotors in in situ vaccines not only solves the limitations of traditional vaccines in antigen enrichment and delivery, but also significantly enhances the anti-tumor immune response through active movement and precise targeting, providing a new solution for cancer treatment.

[0076] (4) The present invention is the first to use an enzyme that degrades DNA or an enzyme that degrades ATP to modify the nanomotor, thereby enhancing the nanomotor's mobility and ability to capture antigens.

[0077] Compared with existing technologies, this solution uses the motility of nanomotors to capture tumor-associated antigens responsively released by the tumor microenvironment, and for the first time realizes an in situ vaccine strategy in which nanomotors enrich tumor-associated antigens and adjuvants (i.e., bacterial outer membrane vesicles OMVs). This innovative technology breaks through the limitations of traditional vaccines, using the active motion ability of nanomotors to accurately capture and enrich tumor antigens and adjuvants, generating in situ vaccines at the tumor site, significantly enhancing antigen presentation efficiency and immune activation capabilities. This solution not only overcomes the challenges of the tumor's immunosuppressive microenvironment, but also provides tumor-associated antigens in situ by inducing immunogenic death of tumor cells, activating a powerful anti-tumor immune response, and providing a new solution for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Prepared for Example 1 D DMSN@ M OMV PFTransmission electron microscopy image (A), Zeta potential diagram (B), release curve of MTO (C), and motility characterization results with and without DNase (D); among them, the left figure in C is the release curve obtained at pH 7.4, and the right figure is the release curve obtained at pH 6.5; three parallel experiments were performed for each sample at each concentration in D, n=3.

[0079] Figure 2 for D DMSN@ M OMV PF Effects on B16F1 cells; A is the cell viability test result, B is the live-dead staining assessment result, C is the cell apoptosis assessment result, and D is the immunofluorescence staining result of CRT and HMGB1.

[0080] Figure 3 for D DMSN@ M OMV PF Quantitative graph of in vitro captured antigen; where A is D DMSN@ M OMV PF Quantitative results of captured proteins; B is D DMSN@ M OMV PF Coomassie blue staining of captured protein; C is D DMSN@ M OMV PF Captured antigen abundance map; D is D DMSN@ M OMV PF Western blot images of captured GP100, TRP 2, and ompA.

[0081] Figure 4 Figure 1 is the result of in vivo efficacy test; A is the tumor growth curve of the mouse subcutaneous melanoma model; B is the H&E staining result of the tumor tissue after treatment; C is the mature DC and tumor-infiltrating T lymphocyte CD8 in the inguinal lymph node + T cells and CD4 + D is the ratio of T cells, and D is the secretion level of inflammatory factors TNF-α and IFN-γ in tumor tissue.

[0082] Figure 5 Figure 3 shows the distal tumor growth curve of a mouse subcutaneous melanoma (B16F1) model (A) and the infiltration of immune cells in the tumor tissue (B).

[0083] Figure 6 In situ vaccine D DMSN@ M OMV PFFigure 3 shows the immune memory effect detection results (A) and tumor volume statistical results (B). DETAILED DESCRIPTION

[0084] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0085] Example 1: In situ vaccine using nanomotor-captured antigens D DMSN@ M OMV PF Preparation

[0086] (1) DNase-immobilized dendritic silica nanoparticles ( D Preparation of DMSN:

[0087] A 25 wt% CTAC solution (4 mL, deionized water), a 3.5 wt% TEA solution (1 mL, deionized water) and ultrapure water (5 mL) were mixed and stirred at 60°C (400 rpm) for 1 h, and then 1.25 mL of TEOS and 8.75 mL of cyclohexane were added to the bottom of the aqueous phase. The mixture was stirred at 60°C and 400 rpm for 12 h. The obtained product was centrifuged at 10,000 rpm for 30 min, and the solid was taken and washed three times with ethanol. The washed solid was calcined at 550°C for 6 h to remove the CTAC template to obtain DMSN nanoparticles (DMSN NPs).

[0088] 0.27 g of DMSN NPs were dispersed in 40 mL of ethanol, 800 μL of APTES was added, stirred and refluxed at 80 ° C overnight, and then centrifuged to obtain DMSN-NH2, which was then dispersed with 27 mL of deionized water to obtain a DMSN-NH2 solution with a concentration of 10 mg / mL. Then, DNase I solution (1 mL, 5 mg / mL) was added dropwise to all the DMSN-NH2 (10 mg / mL) obtained in the previous step and stirred at 4 ° C for 12 h. Finally, centrifuged at 8000 rpm for 10 min and washed three times with pH 7.4, 0.1 M PBS buffer to obtain D DMSN.

[0089] (2) Bacterial outer membrane vesicles loaded with mitoxantrone ( M Preparation of OMVs:

[0090] Attenuated Salmonella VNP20009 (Salmonella typhimurium YS1646, ATCC 202165) was cultured on Luriabroth (LB) agar at 37°C overnight, and then a single colony was inoculated into 50 mL of LB liquid medium and cultured at 37°C and 150 rpm for 12 h. The culture was diluted with fresh LB liquid medium at a volume ratio of 1:50 and cultured at 37°C for another 3 h until the OD of the bacterial culture reached 0. 600 The value reaches about 1.0, which is the logarithmic growth phase. Take 500 mL of bacterial culture, centrifuge at 4000×g for 10 minutes, sterilize and filter with a 0.45μm vacuum filter, and then concentrate the filtrate using an Amicon centrifugal filter with a molecular weight cutoff of 100kDa (Millipore, USA). Then, use an Optima L-80XP ultracentrifuge (Beckman Coulter) at 150000×g for 2 hours at 4°C to obtain OMVs. Resuspend the OMVs in PBS buffer and store at -80°C for further experiments. Preparation M When OMVs were added, 1 mL of MTO (100 μg / mL) and 2 mL of OMVs (25 μg / mL) were mixed and stirred at 37 °C for 4 h. The mixed solution was physically extruded through a 220 nm polycarbonate membrane seven times using an Avanti microextruder to form M OMVs; free MTO was then removed using a 100 kDa ultrafiltration membrane.

[0091] (3) D DMSN@ M Preparation of OMVPF:

[0092] Will D DMSN suspension (1 mL, 1 mg / mL, solvent: PBS) and M OMVs (1 mL, 0.5 mg / mL, the mass of OMVs was determined by BCA protein assay) were mixed under vortex. Afterwards, the mixed solution was physically extruded through a 0.22 μm polycarbonate membrane seven times using an Avanti microextruder to form D DMSN@ M OMVs were collected by centrifugation at 8000 × g for 20 minutes. D DMSN@ M OMVs were resuspended in 1 mL of PBS at 4°C. D DMSN@ M OMV dispersion was used for subsequent experiments. D DMSN@ MThe OMV dispersion was incubated with 0.4 mg of pH-responsive membrane-disrupting peptide (FLEHLIPPHVIHGLVHAIHH-NH2, synthesized by Sangon Biotech) and 1 mL of DSPE-PEG2000-FA (500 μg / mL) for 12 h to form a D DMSN@ M OMV PF .

[0093] The obtained samples were analyzed by transmission electron microscopy (TEM, JEOL) and laser particle size analyzer (Malvern, Zetasizer Nano ZS90). D DMSN@ M OMV PF Perform morphology and charge characterization: Figure 1 A in D DMSN@ M OMV PF The transmission electron microscope image shows M After OMV coating, the product still maintains radial dendrite structure and spherical shape, and forms a core-shell structure, indicating that M OMV is covered in D The outer surface of DMSN; Figure 1 B in the display D DMSN@ M OMV PF The zeta potential is -17.5 mV.

[0094] The suspension was in PBS (pH 7.4 and 6.5). D DMSN@ M OMV PF Incubate in a shaker (100 rpm). At different time points, take 200 μL D DMSN@ M OMV PF The suspension was centrifuged at 10,000 rpm for 20 min, and the supernatant was collected and measured for absorbance at 610 nm using a SpectraMax M5 (Molecular Devices Corp, Los Angeles, CA USA). The initial MTO concentration was determined using the absorbance of the total sample at 610 nm as the denominator to calculate the MTO release rate. Figure 1 Figure C shows that at pH 6.5, about 89.5% of the MTO was removed after 48 hours. D DMSN@ M OMV PF Released.

[0095] To study the chemotaxis of the nanomotors, a Slide-A-Lyzer microdialysis device (20 kD MWCO, ThermoScientific) was placed in one well of a 6-well cell culture plate. Then, 2.5 μmol L-1 salmon sperm DNA (100 μL) was added to the device, and then 20 μL of 10 μg L-1 was added to the well containing 1.6 mL PBS. -1 of D The movement trajectories of the nanomotors were tracked using an inverted optical microscope and analyzed using Image J. Figure 1 D in the proof D The DNA enzyme on DMSN can promote the enhancement of diffusion ability by hydrolyzing DNA.

[0096] Example 2: In situ vaccine using nanomotor-captured antigens D DMSN@ M OMV PF In vitro cytotoxicity and immunogenic death induction

[0097] Mouse melanoma B16F1 cells were cultured in 96-well plates (1×10 6 per well) and different concentrations (0, 1.625, 3.25, 6.5, 12.5, 25, 50, 100 μg mL -1 )of D DMSN@ M OMV PF After 24 h of incubation, cell viability was determined using the CCK-8 assay. Live / dead cell staining: treated and untreated cells were stained with calcein-AM (1 μmol / L) and PI (1 μmol / L) for 15 min at room temperature in the dark, and cell fluorescence images were observed using an inverted fluorescence microscope. Cell apoptosis detection: treated and untreated cells were stained using the Annexin V-FITC / PI apoptosis detection kit (Meilunbio, Dalian, China), and the BD Accuri TM Apoptotic cells were analyzed by C6 flow cytometry. To detect CRT exposure, B16F1 cells were plated at 5×10 4 The cells were cultured in 24-well plates at a density of 100 cells / well for 24 h and then added D DMSN@ M OMV PF(equal to 50 μg / mL DMSN) for 12 h; next, B16F1 cells were washed three times with 4°C pre-cooled PBS and fixed with 4% paraformaldehyde for 10 min; after washing three times with cold PBS, they were incubated with CRT primary antibody (Shanghai, China) for 1 h, washed three times with PBS, and incubated with FITC-conjugated secondary antibody (Shanghai, China) for 30 min; finally, they were stained with Hoechst 33258 (Shanghai, China) and observed under an inverted fluorescence microscope. Detection of HMGB1 release: B16F1 cells were cultured in 24-well plates for 24 h. D DMSN@ M OMV PF (equal to 50μg / mL DMSN) was added for 24h; then, B16F1 cells were washed with cold PBS and fixed with 4% paraformaldehyde for 20 minutes; next, cells were permeabilized with 0.1% v / v TritonX-100 for 10 minutes; nonspecific binding sites were blocked with 5% v / v FBS for 30 minutes, and then incubated with HMGB1 primary antibody at room temperature for 2 hours, and then washed three times with PBS and incubated with FITC-conjugated secondary antibody in the dark for 30 minutes; finally, cells were stained with Hoechst33258 and observed with a fluorescence microscope. A control group was set up, which was a medium without D DMSN@ M OMV PF .

[0098] The results are as follows Figure 2 As shown in Figure A, loading MTO can effectively reduce the viability of B16F1 cells, indicating that MTO can D DMSN@ M OMV PF was released from the cells to show potent cytotoxicity against B16F1 cells. Figure 2 Panel B shows Calcein-AM / PI live / dead cell staining, which shows the same assay results as MTT. Figure 2 C in the figure is the result of cell apoptosis, which further proves that D DMSN@ M OMV PF Can cause significant cell death. Figure 2 D in the figure is the immunofluorescence image of CRT and HMGB1, which further proves that D DMSN@ M OMV PF It can effectively induce immunogenic death of tumor cells in vitro.

[0099] Example 3: In situ vaccine using nanomotor-captured antigens D DMSN@ M OMV PF Antigen capture

[0100] B16F1 cells were cultured at 1×10 4 Cells were seeded at a density of 10 cells / well in 24-well plates and incubated for 24 hours. D DMSN@ M OMV PF (equal to 50 μ g / mL DMSN) treated cells for 24 hours. After 24 hours, the supernatant was collected and the nanoparticles captured by the antigen were separated from free antigen and cell debris by differential centrifugation (with a rotating speed of 1000 rpm for 5 minutes to remove cell debris, and with a rotating speed of 8000 rpm for 5 minutes to collect the nanoparticles bound to the protein) and the amount of protein was determined using the BCA assay. Subsequently, SDS-PAGE gel electrophoresis was carried out to verify that the nanoparticles can capture protein. The collected nanoparticles were heated to denature the protein, and the same amount of protein (20 μ g) was used for electrophoresis. After electrophoresis ended, the gel was stained with Coomassie blue. Finally, the stained gel was decolorized and photographed for analysis.

[0101] Subsequently, the protein bands were enzymatically digested, desalted, and peptide fragments were separated and identified by capillary liquid chromatography tandem mass spectrometry (LC-MS / MS). D DMSN@ M OMV PF The relative abundance of captured proteins was determined by dividing the sample abundance (normalized) value by the B16F1 tumor cell abundance (normalized) for each protein.

[0102] For western blotting, proteins were separated by 10% w / v SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% w / v milk and washed three times with TBST. The membranes were then incubated with anti-GP100 antibody (volume ratio 1:1000), TRP-2 mouse monoclonal antibody (volume ratio 1:1000), or OmpA antibody (volume ratio 1:1000) at 4°C overnight. The membranes were then incubated with HRP-conjugated goat anti-mouse IgG (H+L) (volume ratio 1:1000) at room temperature for 2 hours. Protein bands were detected using a chemiluminescence imaging system.

[0103] The results are as follows Figure 3 As shown in the figure, the dendritic silica nanoparticles without DNase immobilization adsorbed less protein than the dendritic silica nanoparticles with DNase immobilization, which should be attributed to the driving force between DNase and DNA release, which significantly increased the capture of antigens.

[0104] Example 4: In situ vaccine using nanomotor-captured antigens D DMSN@ M OMV PF In vivo efficacy

[0105] 100 μL B16F1 cells (1×10 6 cells / mL) were injected subcutaneously into the back of C57BL / 6 mice to establish a B16F1 tumor-bearing model. When the primary tumor volume reached 100 mm 3 At the same time, the primary tumor was injected with D DMSN@ M OMV PF (equivalent to 50 mg / kg DMSN). On day 16, mice were sacrificed and tumor tissues were collected for photography and H&E staining. Body weight and tumor volume were monitored daily during treatment.

[0106] B16F1 tumor-bearing mice were treated as described above. Seven days after treatment, the mice were euthanized and tumor tissue and inguinal lymph nodes were collected from each group. The tumor tissue and inguinal lymph nodes were minced, filtered through a 300-mesh sieve to obtain single cells, and washed with cold PBS. The cells were centrifuged at 1100 rpm for 5 minutes, resuspended in 100 μL of cold PBS, and labeled with fluorescent antibodies. Flow cytometry was used to detect the presence of mature DCs (CD11c + CD80 + CD86 + ) and tumor-infiltrating T lymphocytes CD8 + T cells (CD3 + CD8 + ) and CD4 + T cells (CD3 + CD4 + To determine the levels of TNF-α and IFN-γ in tumor tissue, tumor tissue was homogenized with ice-cold tissue lysis buffer (Beyotime, China). The supernatant was centrifuged at 8000 × g for 10 minutes and assayed using ELISA kits (biolegenda, San Diego, CA, USA).

[0107] Figure 4 A in the figure is the mouse tumor growth curve. Figure 4 B in the figure is the H&E staining result of the tumor tissue after treatment. Figure 4 C in the figure represents mature DCs in the inguinal lymph nodes and tumor-infiltrating T lymphocytes CD8 + T cells and CD4 + The proportion of T cells, Figure 4 D in the figure represents the secretion of inflammatory factors TNF-α and IFN-γ in tumor tissue. The above results show that the in situ vaccine of nanomotor-captured antigens D DMSN@ M OMV PFIt can effectively inhibit tumor growth and effectively stimulate antigen-specific immune responses.

[0108] Example 5: In situ vaccine using nanomotor-captured antigens D DMSN@ M OMV PF Distal tumor suppressor

[0109] The in vitro efficacy of orthotopic vaccines was investigated using a C57BL / 6 bilateral tumor model. Briefly, B16F1 cells (5 × 10 4 cells / mouse) for primary tumor growth. Four days later, 1×10 4 cells for distant tumor growth. When the primary tumor volume reaches 100 mm 3 When using D DMSN@ M OMV PF The primary tumor was treated with DMSN (50 mg / kg DMSN), while the distal tumor was left untreated. The tumor volume was measured every 2 days to evaluate the therapeutic effect. The mice were killed on the 16th day and the immune cell infiltration was observed. The distal tumor tissue and spleen were sliced ​​and filtered through a 300-mesh sieve to obtain single cells. Anti-CD3-FITC, anti-CD4-PE and anti-CD8-APC antibodies were used to detect CD4 + T cells (CD3 + CD4 + T cells) and CD8 + T cytotoxic T cells (CD3 + CD8 + T cells) were stained and analyzed by flow cytometry.

[0110] Figure 5 A in the figure is the distal tumor growth curve of mice. Figure 5 B in the figure represents CD8 T lymphocytes infiltrating the distal tumor group after treatment. + T cells and CD4 + The results indicate that nanomotors capture antigens for in situ vaccination. D DMSN@ M OMV PF It can significantly inhibit the growth of distal tumors and induce CD8 + T cells and CD4 + T cells are enriched in tumor tissues. The nanovaccine platform of the present invention, when combined with the corresponding antigen, has a significant immunotherapy effect and has a good application prospect.

[0111] Example 6: In situ vaccine using nanomotor-captured antigens D DMSN@ M OMVPF immune memory

[0112] Establish D DMSN@ M OMV PF Secondary B16F1 tumor inoculation model after treatment. First, 5×10 4 B16F1 cells. When the tumor volume reaches 100mm 3 The mice were randomly divided into 6 groups (n=10) and injected intratumorally on days 0, 2, and 4. D DMSN@ M OMV PF On day 40, five mice in each group were sacrificed, and their spleens were removed for analysis of effector memory T cells. The remaining three mice were inoculated with 1×104 B16F1 cells in the left flank. Tumor volume was measured every other day.

[0113] Figure 6 A in the figure is the proportion of effector memory T cells in mouse spleen tissue. Figure 6 B is the growth curve of the tumor after treatment. D DMSN@ M OMV PF It can effectively stimulate the body to produce immune memory effects, effectively inhibit the growth of re-inoculated tumors, and has the function of preventing tumor formation.

[0114] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for constructing an in situ vaccine for tumor antigen capture driven by nanomotor, characterized in that The steps include: (1) Preparation of enzyme-immobilized dendritic silica nanoparticles: A. mixing cetyltrimethylammonium chloride solution, triethylamine solution and water to obtain a mixed solution; B. adding ethyl orthosilicate and cyclohexane to the bottom of the aqueous phase of the mixed solution and stirring, followed by solid-liquid separation, washing the obtained solid, and then removing the template hexadecyltrimethylammonium chloride to obtain porous nanoparticles; C. functionalizing the porous nanoparticles with triethoxysilane to obtain amino-functionalized nanoparticles; D. dropping the enzyme solution into the amino-functionalized nanoparticle solution with stirring, separating the solid and the liquid, and washing the obtained solid to obtain enzyme-immobilized dendritic silica nanoparticles; (2) Preparation of drug-loaded bacterial outer membrane vesicles: The bacterial culture supernatant is centrifuged by ultracentrifugation to obtain bacterial outer membrane vesicles; the drug solution is then mixed with the bacterial outer membrane vesicle solution and extruded through a liposome extruder to remove unloaded drugs, thereby obtaining drug-loaded bacterial outer membrane vesicles; (3) Preparation of in situ vaccines driven by nanomotor-driven tumor antigen capture: A. mixing a dispersion of enzyme-immobilized dendritic silica nanoparticles and drug-loaded bacterial outer membrane vesicles, extruding, and performing solid-liquid separation. The obtained solid is dispersed in a solvent to obtain a suspension. B. The suspension is co-incubated with a pH-responsive membrane-disrupting peptide and a receptor molecule targeting tumor cells to obtain an in situ vaccine that captures tumor antigens driven by nanomotor; The enzyme described in step (1)D is an enzyme that degrades DNA or an enzyme that releases energy.

2. The method for constructing an in situ vaccine for nanomotor-driven tumor antigen capture according to claim 1, characterized in that: The enzyme that degrades DNA is DNase; The energy-releasing enzyme is an ATP-releasing enzyme; The bacteria in step (2) is at least one of Salmonella enterica, lactic acid bacteria, attenuated Salmonella typhimurium, Shigella, Escherichia coli and Bifidobacterium; The drug described in step (2) is an anti-tumor drug; The amino acid sequence of the pH-responsive membrane-disrupting peptide described in step (3)B is as follows: FLEHLIPPHVIHGLVHAIHH-NH2; The receptor molecule targeting tumor cells in step (3)B is DSPE-PEG 2000 -FA.

3. The method for constructing an in situ vaccine for nanomotor-driven tumor antigen capture according to claim 1, characterized in that: In step (1): The hexadecyltrimethylammonium chloride, triethylamine, ethyl silicate and cyclohexane are mixed in a ratio of 1 g: 0.03-0.04 g: 1.0-1.5 mL: 8.5-9.0 mL; The amount of triethoxysilane used is calculated based on 3.0 to 3.5 mL of triethoxysilane per gram of porous nanoparticles; The amount of the enzyme is calculated based on a mass ratio of 5:250 to 300 between the enzyme and the amino-functionalized nanoparticles. The drug in step (2) and the bacterial outer membrane vesicles are mixed in a mass ratio of 1.5 to 2.5:1; The enzyme-immobilized dendritic silica nanoparticles, drug-loaded bacterial outer membrane vesicles, pH-responsive membrane-destroying peptides and tumor cell-targeting receptor molecules described in step (3) are mixed in a mass ratio of 1-3:1:05-1.0:0.8-1.

2.

4. The method for constructing an in situ vaccine for nanomotor-driven tumor antigen capture according to claim 1, characterized in that: The concentration of the hexadecyltrimethylammonium chloride solution in step (1) A is 20-30% wt; The concentration of the triethylamine solution described in step (1) A is 3-4% wt; The concentration of the enzyme solution in step (1)D is 4 to 6 mg / mL; The concentration of the amino-functionalized nanoparticle solution in step (1)D is 8 to 12 mg / mL; The concentration of the drug solution in step (2) is 80 to 120 μg / mL; The concentration of the bacterial outer membrane vesicle solution in step (2) is 20-30 μg / mL; The concentration of the enzyme-immobilized dendritic silica nanoparticle dispersion described in step (3)A is 0.5 to 1.5 mg / mL; The concentration of the drug-loaded bacterial outer membrane vesicles described in step (3)A is 0.4-0.6 mg / mL.

5. The method for constructing an in situ vaccine for nanomotor-driven tumor antigen capture according to claim 1, characterized in that: The stirring conditions described in step (1) A are stirring at 50-70° C. and 300-500 rpm for 30-90 min; The stirring conditions described in step (1) B are stirring at 50-70° C. and 300-500 rpm for 10-15 hours; The solid-liquid separation method in step (1)B is centrifugation; The washing solvent in step (1)B is ethanol; The removal method described in step (1)B is calcination; The stirring condition in step (1) D is stirring at 2-8° C. for 10-16 h; The solid-liquid separation method in step (1)D is centrifugation; The washing solvent in step (1)D is PBS; The ultracentrifugation conditions in step (2) are as follows: centrifugation at 120,000 to 180,000 g for 1 to 3 hours at 2 to 8°C; The mixing condition in step (2) is stirring at 35-40° C. for 3-5 hours; The extrusion described in step (2) used a 220 nm polycarbonate film; The number of extrusions in step (2) is 6 to 8 times; The removal of the unloaded drug in step (2) is achieved by ultrafiltration using a 100 kDa ultrafiltration membrane; The mixing method described in step (3)A is vortex mixing; The extrusion described in step (3)A used a 220 nm polycarbonate film; The number of extrusions in step (3)A is 6 to 8 times; The solid-liquid separation method in step (3)A is centrifugation; The solvent described in step (3) A is PBS; The incubation time described in step (3)B is 10 to 16 hours.

6. The method for constructing an in situ vaccine for nanomotor-driven tumor antigen capture according to claim 5, characterized in that: The stirring conditions described in step (1) A are stirring at 60° C. and 400 rpm for 60 min; The stirring conditions described in step (1)B are stirring at 60°C and 400 rpm for 12 hours; The solid-liquid separation conditions described in step (1) B are centrifugation at 8000-12000 rpm for 20-40 min; The calcination conditions are calcining at 500-600°C for 5-7h; The stirring condition in step (1) D is stirring at 4° C. for 12 h; The solid-liquid separation conditions in step (1) D are centrifugation at 6000-10000 rpm for 5-15 min; The ultracentrifugation conditions described in step (2) are as follows: centrifugation at 150,000 g for 2 h at 4°C; The mixing condition in step (2) is stirring at 37° C. for 4 h; The solid-liquid separation conditions described in step (3) A are centrifugation at 6000-10000 g for 10-30 min; The solvent described in step (3)A is PBS at 2-8°C.

7. The method for constructing an in situ vaccine for nanomotor-driven tumor antigen capture according to claim 1, characterized in that: The specific steps of functionalizing the porous nanoparticles with triethoxysilane in step (1) C are as follows: dispersing the porous nanoparticles in ethanol, adding triethoxysilane, stirring evenly, and then refluxing, and separating the solid and the liquid to obtain solid amino-functionalized nanoparticles; The bacterial supernatant in step (2) is prepared by the following steps: culturing the bacteria to the logarithmic growth phase to obtain a culture solution; and concentrating the liquid obtained by removing the bacterial cells from the culture solution.

8. The method for constructing an in situ vaccine for nanomotor-driven tumor antigen capture according to claim 7, characterized in that: The amount of ethanol used is such that the volume of ethanol is 140 to 150 times the mass of the porous nanoparticles; The reflux condition is reflux at 70-90° C. for 8-16 hours; The specific steps of removing the bacteria are as follows: first centrifugation and then filtering.

9. An in situ vaccine for tumor antigen capture driven by nanomotor, characterized by: It is obtained by the construction method according to any one of claims 1 to 8.

10. Use of the nanomotor-driven in situ vaccine for tumor antigen capture according to claim 9 in the preparation of anti-tumor drugs.

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