Yeast bionic vaccine based on metal organic framework ZIF-8 as well as construction method and application of yeast bionic vaccine

Abstract

The invention belongs to the field of nano biomedicine, and particularly discloses a yeast bionic vaccine based on a metal organic framework ZIF-8 as well as a construction method and application of the yeast bionic vaccine, and the structure of the bionic vaccine is that ZIF-8 loaded with antigen OVA is encapsulated in a yeast shell. The invention relates to preparation and anti-tumor immune response of the bionic vaccine. The yeast shell is obtained by performing acid and alkali treatment and organic solvent washing on saccharomycetes and has the effects of targeting antigen presenting cells and stimulating immune response, and the antigen-loaded metal organic framework ZIF-8 is encapsulated in the yeast shell and has relatively good stability and an effect of continuously releasing antigens. The ZIF-8 is decomposed in antigen presenting cells, and lysosome escape of the antigen is induced. The yeast bionic vaccine provided by the invention can specifically target antigen presenting cells, continuously release tumor antigens in vivo, mediate escape of lysosome of the antigens and stimulate strong anti-tumor immune response, and has a better application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanobiomedicine and tumor immunotherapy, and relates to a yeast bionic vaccine based on a metal organic framework ZIF-8 and a construction method and application thereof, and specifically to the construction of an anti-tumor vaccine using microbial components and a metal organic framework and its products and applications. Background Art

[0002] Traditional tumor vaccines deliver tumor-specific antigens to exert anti-tumor immune responses. However, the efficacy and prognostic effect of activating immune responses using only antigen delivery are limited. In order to induce sustained and extensive anti-tumor immune responses, it is necessary to exert the synergistic effect of tumor-specific antigen delivery and immune response stimulation through drug delivery systems. At the same time, in order to overcome tumor immune tolerance, it is necessary to continuously provide antigens and immune stimulation to the immune system.

[0003] Metal-organic framework materials are crystalline materials with porous structures formed by self-assembly of transition metal ions or metal clusters and organic ligands. Among them, ZIF-8 is a metal-organic framework composed of zinc ions and dimethylimidazole coordination. It has adjustable pore size, large specific surface area, good biocompatibility, high drug loading capacity, and releases drugs in low pH environments. It has good application prospects in anticancer drug delivery. ZIF-8 is stable in neutral or alkaline pH environments, while in acidic pH environments, its dimethylimidazole is protonated, resulting in the release of the coordination effect with zinc ions and the release of drugs.

[0004] ZIF-8 has good biocompatibility and pH responsiveness. It can stably encapsulate proteins through biomimetic mineralization and is a promising carrier for intracellular delivery of biomacromolecules. However, due to the lack of specific targeting, it leads to non-specific absorption of ZIF-8 and dose-dependent cytotoxic side effects. Yeast shell, as a biomimetic carrier of pathogens, can not only be used for antigen presenting cell targeting, but also has obvious advantages in phagocytic specificity and immunostimulation. Its porous shell is conducive to drug loading, but it may also cause unexpected drug leakage, thus limiting the research and application in drug delivery systems.

[0005] The present invention has been found through research that the model antigen OVA is encapsulated in ZIF-8 through bionic mineralization, the nanoparticle size is controllable, the encapsulation rate reaches more than 90%, and acid-responsive drug release is achieved. At the same time, the yeast shell is negatively charged, the ZIF-8 antigen carrier is positively charged, and the electrostatic force is adsorbed and internalized into the yeast shell. The electrostatic force interaction better stabilizes the ZIF-8 antigen carrier inside the yeast shell. Therefore, the bionic vaccine of the present invention has a higher antigen load and physiological stability, and has better acid stability than the carrier of ZIF-8 encapsulated OVA. In addition, the yeast shell has the ability to resist lysosomal acid decomposition, and can realize the sustained release and antigen presentation of antigens in antigen-presenting cells. Compared with the ZIF-8 shell adsorbed on the surface of bacteria, it has a higher antigen encapsulation efficiency and low toxic side effects. Summary of the invention

[0006] The purpose of the present invention is to provide a yeast bionic vaccine based on metal organic framework ZIF-8 and its construction method and application. The vaccine continuously stimulates immune response and releases antigens in vivo, and activates immune response in a fungal infection mode, which is used to strengthen humoral immunity and cellular immunity of tumor vaccines, and induce antigen-specific immune response, so as to construct an efficient tumor vaccine.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] A yeast bionic vaccine based on a metal organic framework ZIF-8. The bionic material yeast shell is used as the coating material of the vaccine delivery system, and the antigen-loaded metal organic framework ZIF-8 nanoparticles are encapsulated inside the yeast shell.

[0009] The method for constructing a yeast biomimetic vaccine based on the metal organic framework ZIF-8 comprises the following steps:

[0010] (1) ZIF-8 encapsulates antigens through biomimetic mineralization to obtain antigen@ZIF-8;

[0011] (2) Yeast is digested with acid and alkali and washed with organic solvent to obtain yeast shell (YS);

[0012] (3) Antigen@ZIF-8 is adsorbed by electrostatic force and enters the yeast shell, achieving the encapsulation of antigen@ZIF-8 inside the yeast shell and obtaining a yeast bionic vaccine based on the metal organic framework ZIF-8.

[0013] In the step (1), the antigen is first mixed with an aqueous solution of 2-methylimidazole, and then an aqueous solution of zinc ions is added to react to obtain antigen@ZIF-8.

[0014] Furthermore, the antigen is first mixed with an aqueous solution of 2-methylimidazole for reaction, and then an aqueous solution of zinc ions is added dropwise, the reaction is stirred, and the precipitate is collected by centrifugation.

[0015] Furthermore, in the step (1), after the precipitate is collected by centrifugation, the precipitate is first washed with anhydrous ethanol, then washed with an SDS aqueous solution, the precipitate is collected by centrifugation, and washed with deionized water to remove excess SDS on the surface of the nanoparticles.

[0016] In the step (2), the yeast is dispersed in a NaOH solution, heated at a constant temperature for reaction, then centrifuged and the supernatant is discarded, washed with ultrapure water, and the precipitate is uniformly dispersed in ultrapure water again, the pH is adjusted with an HCl solution, heated and stirred for reaction, centrifuged, washed with water, the supernatant is discarded, and then washed with isopropanol and acetone in sequence, centrifuged, and the supernatant is discarded.

[0017] Further, the pH was adjusted to 4.5 with HCl solution, and finally the sample was freeze-dried overnight to obtain yeast shells.

[0018] In the step (3), the yeast shell aqueous solution is added to the antigen@ZIF-8 aqueous solution and incubated to obtain the product.

[0019] Furthermore, the yeast shell aqueous solution was incubated at 37° C. with stirring for at least 30 min to allow the yeast shells to fully swell and then be used to coat the metal organic framework ZIF-8 nanoparticles.

[0020] The application of the yeast biomimetic vaccine based on ZIF-8 is used to prepare a delivery system and related preparations that specifically targets antigen presenting cells, continuously releases tumor antigens in vivo, mediates lysosomal escape of antigens and stimulates anti-tumor immune responses.

[0021] Beneficial effects of the present invention:

[0022] 1. The present invention prepares a yeast biomimetic vaccine based on ZIF-8, which not only improves the low targeting of ZIF-8, but also solves the problems of insufficient drug loading and premature leakage of yeast shell. The invention utilizes the characteristics of pH-responsive drug release in low acidic environment of ZIF-8, the endosomal / lysosomal escape mediated by protonation of imidazole, the stable and efficient loading of antigen protein, and the binding of yeast shell with opposite charge through electrostatic force. Through the encapsulation of ZIF-8 by yeast shell, the targeting of antigen presenting cells (APC) is achieved, and the stability of ZIF-8 in vivo is improved.

[0023] 2. The ZIF-8-based yeast bionic vaccine prepared by the present invention uses yeast shells derived from yeast as immune adjuvants to stimulate the body's immune response. Its immune stimulation signal pathway is clear and clear, and it does not contain endotoxins such as LPS. The vaccine is biosafe and can exert a strong and controllable immune stimulation effect in the body.

[0024] 3. The ZIF-8 antigen carrier prepared by the present invention has a selectable tumor antigen loading capacity, can cope with a variety of cancer tumor models and exert antigen-specific immune responses, and the vaccine has a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : Transmission electron microscopy images of ZIF-8 nanoparticles at different molar ratios of zinc ions and dimethylimidazole, and transmission electron microscopy images of OVA@ZIF-8 antigen carriers after adding different masses of model antigen protein OVA;

[0026] in: Figure 1 ac are transmission electron microscopy images of ZIF-8 nanoparticles at different molar ratios of zinc ions and dimethylimidazole; Figure 1 df are transmission electron microscopy images of OVA@ZIF-8 antigen carriers after adding different masses of model antigen protein OVA when the molar ratio of zinc ion to dimethylimidazole was 1:70.

[0027] Figure 2 : The concentration of OVA released from the OVA@ZIF-8 antigen carrier in the incubation supernatant under different pH environments.

[0028] Figure 3 : The relative amount of OVA released in the supernatant of the OVA@ZIF-8@YS biomimetic vaccine at physiological pH for seven days; and the relative amount of OVA released in the supernatant at physiological and acidic pH for 24 hours;

[0029] in: Figure 3 a is the relative content of OVA released in the supernatant of the OVA@ZIF-8@YS bionic vaccine under physiological pH conditions within seven days of incubation;

[0030] Figure 3 b Relative content of OVA released in the supernatant after 24 h of incubation under physiological and acidic pH conditions.

[0031] Figure 4 : Scanning electron microscopy images of yeast shell YS and scanning electron microscopy and transmission electron microscopy images of OVA@ZIF-8 biomimetic vaccine;

[0032] in: Figure 4 a, b are scanning electron microscope images of yeast shell YS at different magnifications; Figure 4 c, d are scanning electron microscopy and transmission electron microscopy images of OVA@ZIF-8@YS bionic vaccine.

[0033] Figure 5 : The stability results of OVA@ZIF-8@YS bionic vaccine in dendritic cells and macrophages for 15 days;

[0034] in: Figure 5 a, b are the results of high-content fluorescence imaging and flow cytometry of OVA@ZIF-8@YS biomimetic vaccine internalized by dendritic cells;

[0035] Figure 5 c, d are the high-content fluorescence imaging and flow cytometry results of OVA@ZIF-8@YS bionic vaccine internalized by macrophages.

[0036] Figure 6 :High-content fluorescence imaging and flow cytometry results of OVA@ZIF-8@YS biomimetic vaccine co-incubated with dendritic cells and macrophages for 8 hours;

[0037] in: Figure 6 a, b are the results of high-content fluorescence imaging and flow cytometry of OVA@ZIF-8@YS biomimetic vaccine internalized by dendritic cells;

[0038] Figure 6 c, d are the results of high-content fluorescence imaging and flow cytometry of OVA@ZIF-8@YS biomimetic vaccine internalized by macrophages;

[0039] Figure 6 e is the high-content fluorescence imaging comparison result of the internalization of OVA@ZIF-8@YS bionic vaccine by non-antigen presenting cells and antigen presenting cells.

[0040] Figure 7 : High-content fluorescence imaging and colocalization coefficient results of OVA@ZIF-8@YS bionic vaccine mediated lysosomal escape in macrophages.

[0041] Figure 8 : The OVA@ZIF-8@YS bionic vaccine stimulates the expression of dendritic cell maturation markers in vitro, as well as a schematic diagram of healthy C57BL / 6 mice inoculated with the OVA@ZIF-8@YS bionic vaccine and the proportion and activation of CD4 and CD8 positive T cells in the spleen T cells of mice after inoculation;

[0042] in: Figure 8 a and b are the OVA@ZIF-8@YS bionic vaccine stimulating dendritic cell maturation in vitro, including the expression of CD80 and CD86 positive dendritic cells and the expression of the specific peptide SIINFEKL-H-2Kb of the model antigen OVA and a class of major histocompatibility antigen complex on dendritic cells; Figure 8c and d are schematic diagrams of healthy C57BL / 6 mice immunized with OVA@ZIF-8@YS bionic vaccine, as well as the proportion and activation of CD4 and CD8 positive T cells in the spleen T cells of mice after vaccination.

[0043] Fig. 9 : Schematic diagram of B16F10-OVA tumor cell inoculation and OVA@ZIF-8@YS biomimetic vaccination, as well as blind tumor growth curves and weight changes of tumor-bearing mice during inoculation with different vaccine preparations, and photos of the quality and morphology of tumor tissues isolated from tumor-bearing mice inoculated with different vaccine preparations;

[0044] in: Fig. 9 a is a schematic diagram of B16F10-OVA tumor cell inoculation and OVA@ZIF-8@YS bionic vaccination; Fig. 9 b and c are the blind tumor growth curves and weight changes of tumor-bearing mice during vaccination with different vaccine preparations; Fig. 9 d, e are photos of the mass and morphology of tumor tissues isolated from tumor-bearing mice vaccinated with different vaccine preparations.

[0045] Fig.10 : Expression of dendritic cell markers in tumor tissues isolated from tumor-bearing mice vaccinated with different vaccine formulations;

[0046] in: Fig.10 a, b are the expression of CD80 and CD86 positive dendritic cells in tumor tissues isolated from tumor-bearing mice vaccinated with different vaccine preparations and the expression of the specific peptide SIINFEKL-H-2Kb of the model antigen OVA and major histocompatibility antigen class I complex on dendritic cells.

[0047] Fig.11 : The proportion and activation of CD4- and CD8-positive T cells in tumor tissues isolated from tumor-bearing mice vaccinated with different vaccine formulations, as well as the activation of regulatory T cells and IFN-γ immunofluorescence sections of tumor tissues;

[0048] in: Fig.11 a, b, c, d are the proportions and activation of CD4 and CD8 positive T cells in tumor tissues isolated from tumor-bearing mice vaccinated with different vaccine preparations; Fig.11 e is the activation of regulatory T cells; Fig.11 f is the IFN-γ immunofluorescence section of tumor tissue.

[0049] Fig.12: Secretion of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interferon-γ (IFN-γ) in tumor tissues isolated from tumor-bearing mice vaccinated with different vaccine formulations;

[0050] in: Fig.12 a is TNF-α, Fig.12 b is IL-6, Fig.12 c is IFN-γ.

[0051] Fig.13 : H&E staining results of major organs isolated from tumor-bearing mice inoculated with different vaccine formulations, including heart, liver, spleen, lung, and kidney.

[0052] Fig.13 This shows that the vaccination of bionic vaccine has no obvious physiological effects or toxic side effects on mice. DETAILED DESCRIPTION

[0053] In order to better illustrate the present invention, the present invention is further described below in conjunction with embodiments.

[0054] Example 1: Preparation and characterization of ZIF-8-based yeast biomimetic vaccine

[0055] (1) 2-MIM (2-methylimidazole) powder (3.15 mmol, 0.2586 g) was accurately weighed on an analytical balance into a stirring bottle with a magnetic rod. After dissolving in 900 μL ultrapure water, 0.50 mg of chicken ovalbumin OVA freeze-dried powder was weighed and added using a 1 / 100000 balance. The mixture was stirred at 800 rpm for 1 h at 30 °C to obtain an OVA / 2-MIM mixed solution. Then, an aqueous zinc nitrate hexahydrate solution (0.045 mmol, 100 μL) was added and stirred vigorously for 10 min. The precipitate was collected by centrifugation at 3500 rpm for 20 min and washed twice with anhydrous ethanol to remove excess ions on the surface of the nanoparticles. In order to remove excess OVA on the surface of the nanoparticles, OVA@ZIF-8 was incubated and washed with a 5% SDS (w / w) aqueous solution at 50 °C for 10 min. The precipitate was collected by centrifugation at 15000 rpm for 20 min and washed three times with deionized water to remove excess SDS on the surface of the nanoparticles. OVA@ZIF-8 was freeze-dried and weighed to determine the amount of product in a single synthesis process and stored in an environment of 4°C.

[0056] (2) Take 5 g of edible yeast and disperse it in 50 mL of a freshly prepared NaOH solution with a concentration of 1 M. Incubate it in an oil bath at a constant temperature of 80°C with stirring for 1.5 h, then centrifuge it at 6000 rpm for 6 min and discard the supernatant. Wash it twice with 25 mL of ultrapure water, and then evenly disperse the precipitate in 25 mL of ultrapure water. Adjust the pH of the suspension sample to 4.5 with a freshly prepared HCl solution with a concentration of 2.5 M. Incubate it in an oil bath at a constant temperature of 55°C with stirring for 0.5 h, then centrifuge it and wash it twice with 25 mL of ultrapure water (6000 rpm, 6 min), discard the supernatant, then wash it 4 times with isopropanol (5 mL / time), and finally wash it twice with acetone (10 mL / time), centrifuge it (6000 rpm, 6 min), mix the sample with a homogenizer (6000 rpm) before each washing, discard the supernatant, freeze-dry the sample overnight to obtain YS, and weigh it to calculate the yield.

[0057] (3) The YS freeze-dried sample was dissolved in ultrapure water and prepared to 10 mg / mL. Ultrasonication was performed for 20 min, and the samples were diluted to 1, 0.33, and 0.2 mg / mL respectively. The OVA@ZIF-8 antigen carrier was dissolved in ultrapure water and prepared to 1 mg / mL. Ultrasonication was performed for 1 min. In order to obtain the internally encapsulated OVA@ZIF-8@YS, 1 mL of YS aqueous solution with different dilution multiples was first incubated at 37 °C with stirring (800 rpm) for 30 min to allow the yeast shell to swell fully. Then, an equal volume of OVA@ZIF-8 antigen carrier aqueous solution was added and incubated for another 30 min. The OVA@ZIF-8@YS was collected by centrifugation (8000 rpm, 8 min) and washed twice with ultrapure water (8000 rpm, 8 min). The obtained samples were freeze-dried and weighed, and the yield, encapsulation efficiency, and drug loading were calculated.

[0058] Figure 1 The results show that different molar ratios of zinc ions and dimethylimidazole, as well as the addition of different masses of OVA, have a significant effect on the particle size of the OVA@ZIF-8 antigen carrier. Too large a particle size may affect the encapsulation efficiency of the OVA@ZIF-8 antigen carrier in the yeast shell YS. The best effect was achieved when the molar ratio of zinc ions and dimethylimidazole was 1:70 and 0.5 mg of OVA was added to prepare the OVA@ZIF-8 antigen carrier.

[0059] Example 2: Determination of encapsulation efficiency and drug loading of yeast biomimetic vaccine based on ZIF-8

[0060] (1) Setting up the OVA standard curve: accurately prepare 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL of OVA suspension, use PBS (pH = 5.5) as the solvent, take 1 mL and dilute it 20 times, then record the UV-visible absorption peak value at 280 nm to draw a standard curve. The specific expression is as follows:

[0061]

[0062]

[0063]

[0064] Dissolve a single synthetic sample in PBS (pH = 5.5) and incubate it at 37 °C for 6 h on a shaker at 100 rpm. After standing for a while, draw 1 mL of the supernatant and dilute it 20 times for UV-visible absorption spectrum test. Set up 3 groups of parallel controls with a scanning band of 400-190 cm -1 According to the UV-visible absorption peak value of the obtained sample, the mass of OVA released from the OVA@ZIF-8 antigen carrier in the supernatant was calculated ( ) and the drug loading capacity of OVA@ZIF-8 antigen carrier ( =7.79%) and encapsulation efficiency ( =90.37%), calculated as follows: the mass of OVA added in a single sample synthesis was 0.5 mg, and the average mass of the freeze-dried samples of OVA@ZIF-8 antigen carriers harvested from a single sample synthesis was 5.8 mg (n=3).

[0065] The single synthesized FITC-OVA@ZIF-8 sample was dissolved in PBS (pH=5.5) and divided into 8 equal parts. The samples were stirred at 37°C and 100 rpm for 0 min, 25 min, 50 min, 1 h, 2 h, 4 h, 6 h, and 8 h, respectively. After the samples were allowed to stand for a period of time, 1 mL of supernatant was aspirated and diluted 100 times for fluorescence spectrophotometry. The excitation wavelength was 488 nm and the emission wavelength region was 505-750 nm. According to the fluorescence value at 515 nm in the supernatant, the mass of FITC-OVA released from the OVA@ZIF-8 antigen carrier in the supernatant was calculated ( ) and its drug loading ( =7.84%) and encapsulation efficiency ( =90.91%).

[0066] (2) Setting up the OVA@ZIF-8 standard curve: Accurately prepare 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mg / mL of OVA@ZIF-8 antigen carrier solution, use ultrapure water as the solvent, dilute 20 times, record the UV-visible absorption peak value at 205 nm, and draw a standard curve. The specific expression is as follows:

[0067]

[0068] Take 1 mL of the synthesized sample supernatant, dilute it 20 times and then perform UV-visible absorption spectrum test with a scanning band of 400-190 cm -1 According to the UV-visible absorption peak value of the obtained supernatant at 205 nm, the OVA@ZIF-8 antigen carrier quality (SQ) in the supernatant and its drug loading in OVA@ZIF-8@YS ( =7.26%) and encapsulation efficiency ( =58.81%):

[0069]

[0070]

[0071]

[0072] in is the result of a single UV spectrum measurement. It refers to the mass of OVA@ZIF-8 antigen carrier in the supernatant (mg). The total volume of the supernatant is 2 mL. The average mass of a single synthesized OVA@ZIF-8@YS freeze-dried sample is 8.1 mg (n=3).

[0073] Example 3: In vitro stability experiment of yeast biomimetic vaccine based on ZIF-8

[0074] (1) Calculation of OVA@ZIF-8 antigen release efficiency under different pH environments: The single synthesized OVA@ZIF-8 samples were dissolved in PBS (pH=5.5) and PBS (pH=7.4), divided into 8 equal portions and added to a 24-well plate, and incubated at 37°C at 100 rpm for 0 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, and 8 h. After incubation, the samples were allowed to stand for a period of time and 1 mL of supernatant was aspirated. After dilution 100 times, the samples were detected by fluorescence spectrophotometer. The pH value of all samples in the PBS (pH=7.4) experimental group was adjusted to 5.5 before detection.

[0075] FITC-OVA standard curve setting for FITC-labeled antigen protein: accurately prepare 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 mg / mL FITC-OVA antigen protein solution, use PBS (pH=5.5) as solvent, dilute 100 times, record the fluorescence emission peak value at 505 nm, and make a standard curve. The specific expression is as follows:

[0076]

[0077] Three groups of parallel controls were set up and the average value was taken. The excitation wavelength was 488 nm, and the emission light receiving wavelength range was 505-750 nm. According to the fluorescence value contained in the supernatant at 515 nm, the mass of FITC-OVA released from FITC-OVA@ZIF-8 in the supernatant was calculated ( ) and drug release efficiency (EE=80.82%):

[0078]

[0079]

[0080]

[0081] (2) In vitro stability test of OVA@ZIF-8@YS under different pH conditions: In order to explore the OVA release of OVA@ZIF-8@YS under different pH conditions (results see Figure 2 and Figure 3 ), single synthesized OVA@ZIF-8@YS (YS:OVA@ZIF-8=1:3) was used for fluorescence spectrophotometry detection. The samples to be tested were suspended in PBS (pH=7.4) and PBS (pH=5.5), respectively, and added to a 6-well plate. Incubated at 37°C in a constant temperature shaker at 100 rpm for the specified time. 1 mL of supernatant was collected at 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h, and measured using a fluorescence spectrophotometer after dilution 50 times.

[0082] OVA@ZIF-8@YS synthesized once was used, with a mass ratio of YS:OVA@ZIF-8=1:3, suspended in PBS (pH=7.4) and added to a 6-well plate. Incubated at 37°C in a constant temperature shaker at 100 rpm for a specified time. 0.1 mL of supernatant was collected at 0d, 1d, 3d, 5d, and 7d. After each supernatant was taken out, an equal amount of PBS (pH=7.4 or pH=5.5) was added and continued to be incubated. After dilution 50 times, the fluorescence spectrophotometer was used for measurement. The cumulative fluorescence intensity released by OVA@ZIF-8@YS ( ) is calculated as follows:

[0083]

[0084] in is the fluorescence intensity of the supernatant obtained at time X (X=1, 3, 5, 7d), is the fluorescence intensity of the supernatant obtained at time 0, The fluorescence intensity of the supernatant of the single-synthesis OVA@ZIF-8@YS is equal to the amount of free OVA@ZIF-8 added in the synthesis.

[0085] (3) Intracellular stability of OVA@ZIF-8@YS within 15 days: In order to observe the intracellular stability of OVA@ZIF-8@YS after internalization by antigen presenting cells, RAW264.7 macrophages or DC 2.4 dendritic cells were inoculated in 96-well plates, with 2×10 4 Cells were cultured in an incubator for 12 h to allow the cells to colonize in a 96-well plate. The culture medium in the cell culture wells was removed, and the concentration of OVA@ZIF8@YS culture medium was prepared at a ratio of 1:20 for the number of cells: number of YS. 100 μL was taken from each well and added to the cell culture wells. The culture medium was incubated at a relative humidity of 95% and CO 2 The cells were cultured in an incubator at a concentration of 5% and a temperature of 37°C for 1, 3, 5, 7, and 15 days. After one day of incubation with the drug, the medium in the cell culture wells was removed and replaced with 100 μL of fresh medium, which was replaced every 2 days. After the specified time of culture, the medium in the corresponding cell culture wells was aspirated and washed once with D-PBS. Hoechst dye was added to the corresponding cell culture wells and incubated on a clean bench for 10 min to stain the cell nuclei. The Hoechst dye in the corresponding cell culture wells was aspirated and washed once with D-PBS. LysoTracker was added ® The Red dye was incubated in the incubator for 10 min to stain the phagosomes, the dye in the corresponding cell culture wells was aspirated, and washed once with D-PBS, and then added with D-PBS again, and observed in a high-content fluorescence imaging system. The excitation light of FITC is 460-550nm, and the emission light is 525nm; the excitation light of Hoechst is 350-405nm, and the emission light is 460nm; LysoTracker ® The excitation light for Red is 550-580nm, the emission light is 590nm, the camera height is 2μm, and the exposure time is 50ms.

[0086] In order to detect the stability of fluorescence intensity of OVA@ZIF-8@YS in antigen presenting cells, RAW264.7 macrophages or DC 2.4 dendritic cells were seeded in 6-well plates, with 2.5×10 5Cells were cultured in an incubator for 18 hours. The culture medium in the cell culture wells was removed, and the culture medium concentration of OVA@ZIF-8@YS was prepared at a ratio of 1:20 for the number of cells: the number of YS. 1 mL was added to each well of the cell culture wells and cultured in an incubator for 1, 3, 5, 7, and 15 days. After 1 day of incubation with the drug, the culture medium in the cell culture wells was removed and replaced with 1 mL of fresh culture medium, and replaced every 2 days. After the specified time of culture was completed, the culture medium in the corresponding cell culture wells was aspirated and washed once with 1 mL of D-PBS. 1 mL of D-PBS was added again and RAW264.7 macrophages or DC 2.4 dendritic cells in the cell culture wells at the corresponding time were scraped with a cell scraper and collected in a 1.5 mL centrifuge tube. The tube was centrifuged at 37°C (1000 rpm, 5 min), and gently blown up with 1 mL of D-PBS and washed once. After centrifugation at 37°C (1000 rpm, 5 min), 300 μL of D-PBS was added to gently blow up RAW264.7 macrophages or DC 2.4 dendritic cells, placed on an ice box, filtered through a 300-mesh screen, and added to a flow tube for flow cytometry testing. The excitation light was 488 nm, the FITC channel (505-545 nm) was selected for reception, and the total number of cells received was 5×10 4 The flow cytometry results were processed by FlowJo software. Figure 5 The OVA antigen carried by the bionic vaccine was still located in the cells after 15 days of incubation with antigen-presenting cells, which means that the intracellular stability of the bionic vaccine can continuously release OVA into the antigen-presenting cells.

[0087] Example 4: Antigen presenting cell specific targeting experiment of yeast biomimetic vaccine based on ZIF-8

[0088] (1) In order to observe the changes of OVA@ZIF-8@YS internalization over time, RAW 264.7 macrophages or DC 2.4 dendritic cells were seeded in 96-well plates. Cells were cultured in an incubator at a relative humidity of 95%, a CO2 concentration of 5%, and a temperature of 37°C for 12 h. The culture medium in each well was removed, and a cell culture medium dispersion solution of OVA@ZIF-8@YS was prepared at a ratio of cell number: YS number = 1:20. 100 μL per well was added to the cell seeding wells and cultured in an incubator at a relative humidity of 95%, a CO2 concentration of 5%, and a temperature of 37°C for 0.5, 1, 2, 4, 6, and 8 h.

[0089] After the designated time of incubation, the medium in the corresponding cell-seeding wells was aspirated, and the cells were washed once with D-PBS and gently blown to remove the uninternalized OVA@ZIF-8@YS. The pre-made LysoTracker ® ​​Red dye was added to the corresponding cell-seeding wells and incubated in an incubator at a relative humidity of 95%, a CO2 concentration of 5%, and a temperature of 37°C for 10 min to stain the phagosomes. The Lyso Tracker ® Red dye was added and washed once with D-PBS; pre-made Hoechst dye was added and incubated on a clean bench for 10 min to stain the cell nucleus, and the cells were washed once with D-PBS, and D-PBS was added again for observation in a high-content fluorescence imaging system.

[0090] For quantitative analysis of the cellular internalization of OVA@ZIF-8@YS over time, RAW 264.7 macrophages or DC 2.4 dendritic cells were seeded in 6-well plates. The cells were cultured under the same conditions for 12 h. OVA@ZIF-8@YS concentrations were prepared in the same ratio and cultured with cells for 0, 0.5, 1, 2, 4, and 8 h. Wash once with D-PBS and gently blow to remove the uninternalized OVA@ZIF-8@YS. D-PBS was added again and the cells in the cell seeding wells at the corresponding time were scraped with a cell scraper and collected in a 1.5 mL centrifuge tube. Centrifuged at 37°C (1000 rpm, 5 min), gently blown up with D-PBS and washed once. After centrifugation at 37°C (1000 rpm, 5 min), 300 μL of D-PBS was added to gently blow up the cells and placed on an ice box for flow cytometry detection.

[0091] (2) In order to observe the cellular internalization degree of OVA@ZIF-8@YS by HeLa cancer cells, L02 hepatocytes, RAW264.7 macrophages and DC 2.4 dendritic cells, the four types of cells were seeded in 96-well plates, with 2×10 cells per well. 4 cells, cultured in an incubator for 12 hours, and diluted OVA@ZIF-8@YS with the culture medium of the corresponding cells at a ratio of 1:20 for the number of cells: number of YS. 100 μL per well was added to the cell culture wells for drug incubation, and cultured in an incubator for 0, 8, and 24 hours. After the specified incubation time, the culture medium in the corresponding cell culture wells was aspirated and washed 1-2 times with D-PBS or PBS, and gently blown to remove the uninternalized OVA@ZIF-8@YS, and Hoechst and LysoTracker were used respectively. ®Red was used to stain the cell nucleus and lysosomes, and the cells were observed in a high-content fluorescence imaging system. Hela cancer cells, L02 hepatocytes, RAW264.7 macrophages, and DC 2.4 dendritic cells were inoculated in 24-well plates, with 1×10 5 Cells were cultured in an incubator for 12 h. OVA@ZIF-8@YS was diluted with the culture medium of the corresponding cells at a ratio of 1:20 for the number of cells and the number of YS. 400 μL per well was added to the cell culture wells for incubation for 0, 8, and 24 h. The culture medium in the corresponding cell culture wells was aspirated and washed 1-2 times with D-PBS or PBS. Hela cancer cells and L02 hepatocytes were digested with trypsin, and RAW264.7 macrophages and DC 2.4 dendritic cells were collected and washed for flow cytometry.

[0092] (3) To determine the lysosomal escape of OVA@ZIF-8@YS in RAW 264.7 macrophages, RAW 264.7 macrophages were seeded in 6-well plates. Cells were cultured in an incubator at a relative humidity of 95%, a CO2 concentration of 5%, and a temperature of 37°C for 18 h. The culture medium in each well was removed, and a cell culture medium dispersion solution of OVA@ZIF-8@YS was prepared at a ratio of cell number: YS number = 1:20. 1 mL was added to the cell planting wells for each well, and cultured in an incubator at a relative humidity of 95%, a CO2 concentration of 5%, and a temperature of 37°C for 2 or 8 h.

[0093] Aspirate the medium in the corresponding cell-seeding wells, wash once with D-PBS, and gently blow to remove uninternalized free OVA, OVA@ZIF-8, OVA@ZIF-8@YS, and YS. Add pre-made Lyso Tracker® Red dye to the corresponding cell-seeding wells and incubate in an incubator with a relative humidity of 95%, a CO2 concentration of 5%, and a temperature of 37°C for 10 min to stain the phagosomes. Aspirate the Lyso Tracker® Red dye in the corresponding cell-seeding wells. ® Red dye was added and washed once with D-PBS; pre-made Hoechst dye was added and incubated on a clean bench for 10 min to stain the nuclei of RAW 264.7 macrophages, and the cells were washed once with D-PBS, and D-PBS was added again for observation in a high-content fluorescence imaging system.

[0094] Results Figure 6 Since yeast shell YS can be specifically internalized by antigen-presenting cells, while non-antigen-presenting cells cannot internalize yeast shell YS, the synthesized OVA@ZIF-8@YS bionic vaccine also produced an internalization effect in macrophages and dendritic cells.

[0095] Figure 7 These results indicate that OVA@ZIF-8@YS decomposes under lysosomal acidic conditions and induces lysosomal escape.

[0096] Example 5: Experiment on the stimulation of dendritic cell maturation by yeast biomimetic vaccine based on ZIF-8

[0097] (1) In vitro dendritic cell maturation assay and cytokine detection: Dendritic cells DC 2.4 were seeded in 6-well plates (2.5×10 5 The cells were incubated for 12 h. The culture medium was supplemented with PBS, free OVA, OVA@ZIF-8, and OVA@ZIF-8@YS (cell:YS particles = 1:20) for 4 h. The supernatant was removed and replaced with fresh medium and cultured for another 24 h. The culture supernatant was collected for ELISA detection of TNF-α, IL-1β, IL-6, and IL-12. DC 2.4 was collected and incubated with RPMI-1640 containing 2% FBS for 10 min, stained with mouse FITC-labeled anti-CD11c antibody, PE-labeled CD86 antibody, and APC-labeled CD80 antibody at 4 °C for 30 min, and washed for flow cytometry detection.

[0098] (2) In vivo detection of dendritic cell-induced T cell activation: A total of 20 healthy C57BL / 6J mice were selected and randomly divided into 4 groups of equal number. PBS, free OVA, OVA@ZIF-8, and OVA@ZIF-8@YS were subcutaneously inoculated three times, with equal amounts of OVA, 100 μL each time, and the interval between each inoculation was 7 days. Seven days after the last immunization, the mice in each group were euthanized, the spleens were separated, ground in a 70-mesh filter, and the red blood cells were lysed to obtain a mouse spleen single cell suspension, which was dispersed into 10 7 After incubating a single-cell suspension of 100 μL / mL with RPMI-1640 containing 2% FBS for 10 minutes, 100 μL of the single-cell suspension was stained with 5 μL of PE-labeled anti-CD3 antibody, FITC-labeled anti-CD4 antibody, and APC-labeled anti-CD8 antibody at 4°C for 30 minutes and then washed for flow cytometry detection.

[0099] Figure 8 The results showed that the OVA@ZIF-8@YS biomimetic vaccine effectively stimulated dendritic cells to express the mature marker CD80 / 86. In addition, dendritic cells co-incubated with the biomimetic vaccine also expressed the MHC-I specific antigen peptide SIINFEKL, which is a marker for OVA to escape from lysosomes and produce antigen cross-presentation by dendritic cells. After inoculation of mice, the biomimetic vaccine effectively activated tumor-specific cytotoxic T cells in vivo.

[0100] Example 6: Yeast biomimetic vaccine based on ZIF-8 induces anti-tumor immune response in vivo

[0101] according to Fig. 9 A total of 20 mice bearing melanoma B16F10-OVA were randomly divided into 4 groups of equal number and subcutaneously inoculated three times with PBS, free OVA, OVA@ZIF-8, and OVA@ZIF-8@YS, with equal amounts of OVA, 100 μL each time, and an interval of 7 days between each inoculation. Seven days after the last immunization, the melanoma-bearing mice were euthanized, and tumor tissues and inguinal lymph nodes were harvested and treated with collagenase IV (1 mg mL -1 ), hyaluronidase (0.1 mg mL -1 ) and DNase I (0.2 mg mL -1 ) in RPMI-1640 (2% FBS) for 3 h. After lysing the red blood cells and incubating with RPMI-1640 containing 2% FBS for 10 min, the cell suspension (n = 5) was stained with mouse PE-labeled anti-CD3 antibody, FITC-labeled anti-CD4 antibody, APC-labeled anti-CD8 antibody, PE-labeled anti-CD25 antibody, and APC-labeled anti-Foxp3 antibody at 4°C for 30 min. The Foxp3 transcription factor staining kit was used for cell fixation and permeabilization, and then stained with APC-labeled anti-Foxp3 antibody. The inguinal lymph nodes were ground, filtered, and a single-cell suspension of lymphocytes was obtained. After incubation with RPMI-1640 containing 2% FBS for 10 min, the lymphocytes (n = 5) were stained with mouse FITC-labeled anti-CD11c antibody, PE The cells were stained with anti-CD86 antibody labeled with APC and anti-CD80 antibody labeled with APC at 4°C for 30 min and then detected by flow cytometry after washing. The contents of TNF-α, IFN-γ and IL-6 in tumor tissues were detected by enzyme-linked immunosorbent assay kit (Elerite Bio, Wuhan, China). The heart, liver, spleen, lung, kidney and tumor of mice were isolated and immersed in 4% paraformaldehyde solution for 24 h. After fixation for 24 h, the mouse tissues were embedded in paraffin and analyzed by H&E staining and immunofluorescence staining.

[0102] Fig. 9 The results showed that the bionic vaccine effectively inhibited the growth of B16F10-OVA tumors in vivo and had no obvious toxic side effects on mice.

[0103] Fig.10The results showed that mice vaccinated with OVA@ZIF-8@YS biomimetic vaccine produced obvious dendritic cell maturation and antigen cross-presentation, corresponding to the expression of maturation markers CD80 / 86 and MHC-I specific antigen peptide SIINFEKL.

[0104] Fig.11 , 12 and 13 showed that mice vaccinated with OVA@ZIF-8@YS bionic vaccine produced effective antigen-specific anti-tumor immune response without obvious toxic side effects.

[0105] The results of Examples 4 to 6 above demonstrate that the anti-tumor vaccine of the present invention specifically targets antigen presenting cells and induces maturation of dendritic cells and activation of T cells. The anti-tumor vaccine continuously releases tumor antigens and stimulates immune response, and effectively inhibits tumor growth.

[0106] The above embodiments are preferred implementation modes of the present invention, which are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. It should be understood that there are many alternative solutions within the scope of the principle of the present invention, which do not affect the essential content of the present invention.

Claims

1. A yeast bionic vaccine based on metal organic framework ZIF-8, characterized in that: The biomimetic material yeast shell is used as the coating material of the vaccine delivery system, and the antigen-loaded metal organic framework ZIF-8 nanoparticles are encapsulated inside the yeast shell.

2. The method for constructing a yeast biomimetic vaccine based on the metal organic framework ZIF-8 according to claim 1, characterized in that: The following steps are involved: (1) ZIF-8 encapsulates antigens through biomimetic mineralization to obtain antigen@ZIF-8; (2) The yeast is digested with acid and alkali and washed with an organic solvent to obtain a yeast shell; (3) Antigen@ZIF-8 is adsorbed by electrostatic force and enters the yeast shell, achieving the encapsulation of antigen@ZIF-8 inside the yeast shell and obtaining a yeast bionic vaccine based on the metal organic framework ZIF-8.

3. The construction method according to claim 2, characterized in that: In the step (1), the antigen is first mixed with an aqueous solution of 2-methylimidazole, and then an aqueous solution of zinc ions is added to react to obtain antigen@ZIF-8.

4. The construction method according to claim 3, characterized in that: In the step (1), the antigen is first mixed with an aqueous solution of 2-methylimidazole for reaction, and then an aqueous solution of zinc ions is added dropwise, stirred for reaction, and the precipitate is collected by centrifugation.

5. The construction method according to claim 4, characterized in that: In the step (1), after the precipitate is collected by centrifugation, the precipitate is first washed with anhydrous ethanol, then washed with an SDS aqueous solution, the precipitate is collected by centrifugation, and then washed with deionized water to remove excess SDS on the surface of the nanoparticles.

6. The construction method according to claim 2, characterized in that: In the step (2), the yeast is dispersed in a NaOH solution, heated at a constant temperature for reaction, then centrifuged and the supernatant is discarded, washed with ultrapure water, precipitated, and uniformly dispersed in ultrapure water again, the pH is adjusted with an HCl solution, heated and stirred for reaction, centrifuged, washed with water, the supernatant is discarded, and then washed with isopropanol and acetone in sequence, centrifuged, and the supernatant is discarded.

7. The construction method according to claim 6, characterized in that: In the step (2), the pH is adjusted to 4.5 with HCl solution, and finally the sample is freeze-dried overnight to obtain yeast shells.

8. The construction method according to claim 2, characterized in that: In the step (3), the yeast shell aqueous solution is added to the antigen@ZIF-8 aqueous solution and incubated to obtain the product.

9. The construction method according to claim 8, characterized in that: The yeast shell aqueous solution was incubated with stirring at 37°C for at least 30 min to allow the yeast shells to fully swell and then used to coat the metal organic framework ZIF-8 nanoparticles.

10. The use of the yeast biomimetic vaccine based on the metal organic framework ZIF-8 according to claim 1, characterized in that: Used to prepare vaccine delivery systems and related preparations that specifically target antigen-presenting cells, continuously release tumor antigens in vivo, mediate lysosomal escape of antigens and stimulate anti-tumor immune responses.

Images