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Physicochemical characterisation

The nanoadjuvants were observed using a field emission scanning electron microscope (FE-SEM, JEOL) after being coated with platinum and using transmission electron microscope (TEM, JEOL). The hydrodynamic diameter of nanoadjuvants was analysed by a dynamic light scattering photometer (DLS-8000HAL, Otsuka Electronics). The phases of nanoadjuvants were analysed using a powder X-ray diffractometer employing CuKα X-ray (Model RINT 2500, Rigaku). The zeta potential of nanoadjuvants was analysed using a Delta Nano C Particle Analyzer (Beckman Coulter Inc, America) by dispersing particles in calcium and magnesium – free phosphate – buffered saline (PBS(−)). The nitrogen gas (N2) adsorption-desorption isotherm of nanoadjuvants was measured by a surface area and porosity analyser (TriStar II, Micromeritics, America) and the BET specific surface areas and pore size distributions were calculated subsequently.

Nanoadjuvants synthesis

MOF, MOF-gated MOF, MS and MOF-gated MS were synthesised in the preliminary experiment as follows. MOF was synthesised by mixing 80 μL of Zn(NO3)2·6H2O (Wako) solution (×1.0, 0.69 M), 400 μL of water and 800 μL of 2-methylimidazole (Wako) solution (×1.0, 3.13 M) with sonication for 20 min in ice. The obtained products were centrifuged, washed with ultra-pure water, and dispersed in solution or freeze-dried. MOF-gated MOF was synthesised by mixing 400 μL of MOF core suspension in aqueous solution at a concentration of 6 mg/mL, 80 μL of Zn(NO3)2·6H2O solution (×0.2, 0.138 M; ×0.4, 0.276 M; ×0.5, 0.345 M) and 800 μL of 2-methylimidazole solution (×0.2, 0.626 M; ×0.4, 1.252 M; ×0.5, 1.565 M) with sonication for 20 min in ice. The obtained products were centrifuged, washed with water, and dispersed in solution or freeze-dried.

MS were synthesised using a soft-templating method39. Typically, hexadecyltrimethylammonium p-toluenesulfonate (CTAT, Sigma-Aldrich) and triethanolamine (TEA, Sigma-Aldrich) were added into ultrapure water with stirring at 70 °C and tetraethoxysilane (TEOS, Wako, Japan) was slowly added. The molar ratio of the reaction mixture was 1.00 TEOS: 0.06 CTAT: 0.026 TEA: 80 H2O, respectively. The reaction mixture was continuously stirred for 2 h to obtain a precipitate. The obtained product was centrifuged, washed with ultrapure water/ethanol, dried and heat-treated at 550 °C for 5 h. MOF-gated MS was synthesised by mixing 400 μL of MS suspensions (0.033 M; 0.100 M; 0.166 M), 80 μL of Zn(NO3)2·6H2O solution (×0.1, 0.069 M; ×0.3, 0.207 M; ×0.4, 0.276 M; ×0.5, 0.345 M; ×0.6, 0.414 M) and 800 μL of 2-methylimidazole solution (×0.1, 0.313 M; ×0.3, 0.939 M; ×0.4, 1.252 M; ×0.5, 1.565 M; ×0.6, 1.878 M), respectively. Then, the products were centrifuged, washed with water, and dispersed in solution or freeze-dried.

Encapsulation of model antigens and immunopotentiators into nanoadjuvants

In the preliminary experiments, Ovalbumin (OVA, Sigma-Aldrich) as a model antigen was encapsulated into MOF to prepare OVAinMOF by mixing 400 μL of OVA aqueous solution (1 mg/mL, 5 mg/mL and 25 mg/mL), 80 μL of Zn(NO3)2·6H2O solution (×1.0, 0.69 M) and 800 μL of 2-methylimidazole solution (×1.0, 3.13 M) with sonication for 20 min in ice followed by being centrifuged, washed with water and dispersed in solution or freeze-dried. In addition, polyinosinic – polycytidylic acid (polyIC, InvivoGen) as an immunopotentiator was encapsulated into MOF using 400 μL of polyIC aqueous solution (1 mg/mL, 5 mg/mL and 25 mg/mL) instead of the OVA aqueous solution.

In the subsequent experiments, MOF-gated MS with and without encapsulating biomolecules were synthesised as follows. Typically, MOF-gated MS with and without encapsulating OVA were synthesised by mixing 600 μL of MS aqueous suspensions with and without OVA, 80 μL of Zn(NO3)2·6H2O solution (×0.2, 0.138 M) and 600 μL of 2-methylimidazole solution (×0.2, 0.626 M) with sonication for 20 min in ice followed by being centrifuged, washed with water and dispersed in solution or freeze-dried. The samples were named MS@(OVAinMOF) and MS@MOF, respectively. Also, MS@(FerritininMOF) was prepared by the same method using ferritin instead of OVA. Then, (MS@OVAinMOF)@(polyICinMOF), (MS@OVAinMOF)@(anti-CTLA4inMOF) and (MS@OVAinMOF)@MOF were prepared by mixing 600 μL of MS@(OVAinMOF) aqueous suspensions with and without polyIC or anti-CTLA4, 80 μL of Zn(NO3)2·6H2O solution (×0.2, 0.138 M) and 600 μL of 2-methylimidazole solution (×0.2, 0.626 M) with sonication for 20 min in ice followed by being centrifuged, washed with water, and dispersed in solution or freeze-dried.

Quantitative approach of particles mass and biomolecules amounts

The mass of MS and MOF in MOF-gated MS is calculated by measuring average weight before and after synthesis reaction, with the exclusion of the dissolution of MS itself (n = 3). The mass of MOF is calculated by measuring average weight before and after synthesis reaction (n = 3). The concentrations of OVA, ferritin and anti-CTLA4 antibodies in solutions before and after loading are determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc.). The concentrations of polyIC before and after loading are measured using an ultraviolet-visible spectrophotometer (V-550, JASCO). The encapsulation efficiencies of biomolecules (OVA, ferritin, polyIC and anti-CTLA4, etc.) are calculated by the following formula, respectively: biomolecule encapsulation efficiency = (Initial biomolecule concentration−biomolecule concentration after encapsulation)/Initial biomolecule concentration × 100%. The standard solutions of biomolecules, including OVA, ferritin, anti-CTLA4 antibodies and polyIC are obtained in the same concentration of 2-methylimidazole solution with synthesis parameter.

Degradation of nanoadjuvants associated with release of biomolecules

MOF-gated MS or MOF with and without encapsulating OVA were synthesised. The final mass ratios of MS:MOF in MS@MOF and MS:MOF:OVA in MS@(OVAinMOF) are about 2.4:0.6 and 2.4: 0.6: 1, respectively. The final mass ratios of MOF:OVA in OVAinMOF are about 3: 1. In contrast, OVAonMS was prepared by mixing OVA solution and MS particles with a MS:OVA mass ratio of 3:1. Degradation of MS, MS@MOF, MOF, OVAonMS, MS@(OVAinMOF) and OVAinMOF samples contained in a bag of dialysis membrane in an acetate buffer (pH = 5) or a Tris-HCl buffer (pH = 7.4) at a particles-to-buffer ratio of 1 mg/mL was quantitatively analysed after incubation at 37 °C by measuring Si and Zn using an inductively coupled plasma atomic emission spectrometer (ICP-AES: SPS7800, Seiko Instruments). The OVA release was determined in an acetate buffer (pH = 5) or a Tris-HCl buffer (pH = 7.4) at a particles-to-buffer ratio of 3 mg/mL at 37 °C.

The MS@(polyICinMOF) and polyICinMOF samples were synthesised by the method same as that for the above MS@(OVAinMOF) and OVAinMOF samples except that the mass ratios of MS:MOF:polyIC and MOF:polyIC are 2.4:0.6:0.5 and 3:0.5, respectively. The polyIConMS was prepared by mixing polyIC solution and MS particles with a MS:polyIC mass ratio of about 3:0.5. The polyIC release was determined in an acetate buffer or a Tris-HCl buffer at a particles-to-buffer ratio of 1 mg/mL at 37 °C.

In addition, Tris-HCl buffer supplemented with 10% serum was used as a third type of media to test the degradation of nanoadjuvants associated with release of molecules using same protocol as those for an acetate buffer or a Tris-HCl buffer. In the serum solution, ferritin was used as a model biomolecule, due to the overlap of OVA and serum in spectra. The release of ferritin was quantitatively analysed by measuring Fe using ICP-AES. The release of polyIC in serum solution was tested using a StrandBriteTM Green Fluorimetric RNA Quantitation Kit (AAT Bioquest).

The standard solutions of biomolecules for release experiments, including OVA, ferritin and polyIC, are obtained in acetate buffer, Tris-HCl buffer or Tris-HCl buffer supplemented with 10% serum, according to the corresponding experimental parameters.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

OVA-loaded nanoadjuvants, including OVAonMS, MS@(OVAinMOF) and OVAinMOF, were dispersed in PBS(−) with the final concentration of 200 ng/μL OVA and 800 ng/μL particles, respectively. Free OVA was used as control. To prepare the supernatant samples, OVA-loaded nanoadjuvants were suspended in PBS(−) for 1 h and centrifuged at 13,000 rpm for 10 min. Then, OVA-loaded nanoadjuvants or supernatant samples were mixed with 2× SDS–PAGE sample buffer, incubated at 50 °C for 10 min, loaded into the gel and subjected to electrophoresis at 30 mA for 70 min running with 1× Tris-Glycine-SDS buffer according to the manufacturer’s instructions. The gels were visualised by staining with Rapid Stain Coomassie Brilliant Blue kit.

Cellular uptake and activation of dendritic cells in vitro

Bone marrow derived dendritic cells (BMDCs) were obtained from mice femurs40. After removing red blood cells, I-A/I-E and phycoerythrin-conjugated anti-CD4, CD8 expressing cells, the left cells were cultured in RPMI 1640 (Gibco) containing 10% fetal bovine serum and 20 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF, Bioreagent). The BMDCs were collected on day 9. In all, 2 × 105 BMDCs were precultured in glass bottom dish or 96-well plate for 6 h. Nanoadjuvants prepared using OVA or fluorescein-conjugated OVA (fOVA, Life technologies), where they were OVAonMS, MS@(OVAinMOF), OVAinMOF, fOVAonMS, MS@(fOVAinMOF) and fOVAinMOF, were added to the BMDCs culture media at a particle concentration of 30 μg/mL and a OVA or fOVA concentration of 5 μg/mL. After overnight culture, the BMDCs were stained with Hoechst (Thermo Fisher) for cell nuclei and observed by a confocal laser microscope (Leica). The quantitative analysis of cellular uptake fluorescence images was calculated using image J software. TNF-α and IL-1β in the supernatant were quantified using mouse ELISA kit (BD Biosciences) according to the manufacturer’s instructions. To further measure activation of BMDCs, 2 × 106 BMDCs were cocultured in 24-well plate with free OVA, OVAonMS, MS@(OVAinMOF) and OVAinMOF at a particle concentration of 30 μg/mL and a OVA concentration of 5 μg/mL, respectively. After 3 days’ culture, the BMDCs were collected using Trypsin-EDTA, blocked with anti-CD16/CD32 Ab (2.4G2, BioLegend) with 1/100 dilution and stained with anti-mouse OVA257-264 (SIINFEKL) peptide bound to H-2Kb Ab, anti-mouse MHC II (I-A/I-E) Ab, anti-mouse CD80 Ab, anti-mouse CD40 Ab and anti-mouse CD197(CCR7) Ab (BioLegend) with 1/50 dilution. Flow cytometry was carried out using FACSAria (BD Bioscience, USA). For all flow cytometry experiments, 1–3 million cells per sample were collected for staining. Among them, at least 10–50 thousand cells per sample were used for flow cytometry analysis. Flowjo software was used to analyse the flow cytometry data.

In addition, to compare the difference between adsorption and encapsulation of fOVA or OVA, fOVA- or OVA- adsorbing MOF (MOF-ad) and fOVA- or OVA- encapsulating MOF by coprecipitation (MOF-en) were prepared, and added to the BMDCs culture at a particle concentration of 25 μg/mL and a OVA or F-OVA concentration of 5 μg/mL.

Antigen retention at the injection sites and particle distribution in vivo

Alex Fluor 647-conjugated OVA (A647-OVA, Molecular Probes) or MS@(A647-OVAinMOF) was injected into the flank of the female C57Bl/6J mice (n = 3, every group; CLEA Inc.) at a A647-OVA dose of 100 μg/mouse and particle dose of 600 μg/mouse in 100 μL saline. At 6 h, 1 day and 3 day, the mice were observed using IVIS imaging system with excitation wavelength of 580 nm and emission wavelength of 680 nm. To clearly see the body distribution of nanoparticles, the main organs (nearby draining lymph node, spleen, lung, heart, kidney and liver) of mice were collected and observed using IVIS imaging system at day 1. Living image software was used to analyse the data. Moreover, ICP-AES measurement was used to quantitatively analyse the targeting distribution of nanoparticles (Si and Zn content) in nearby draining lymph node.

APCs-mediated delivery to lymph nodes and cross-presentation of OVA in vivo

Female C57Bl/6J mice (3 mice, every group; CLEA Inc.) were immunised by injecting fOVA, fOVAonMS, MS@(fOVAinMOF) and fOVAinMOF subcutaneously into the left flank at particles and fOVA doses of 600 μg/mouse and 100 μg/mouse, respectively. The immunised mice were killed 16 h later, and the nearby draining lymph node was collected. The cryostat sections of lymph nodes were prepared, stained with DAPI and observed using a fluorescence microscope (Olympus BX51) with a highly-sensitive camera (Olympus DP74). Fluorescent images were acquired under identical parameter settings. The quantitative analysis of fluorescent images was calculated using image J software. Moreover, the draining lymph node was collected, milled, vortexed and filtered through a 40-μm cell strainer to obtain single-cell suspension. The single-cell suspension was washed with PBS(−) containing 0.5% bovine serum albumin (BSA). Non-specific staining was prevented by blocking the cells with anti-CD16/CD32 Ab (2.4G2, BioLegend) with 1/100 dilution. The cells were stained for 30 min with anti-mouse CD11c Ab and anti-mouse OVA257-264 (SIINFEKL) peptide bound to H-2Kb Ab (BioLegend) with 1/50 dilution. Flow cytometry was performed using FACSAria (BD Bioscience, USA).

Prophylactic cancer immunotherapy

Twenty eight female C57Bl/6J mice (7 mice/group; 6 weeks old, CLEA Inc.) were divided into the following four groups: 1# saline group; 2# free OVA-anti-CTLA4 group (OVA, 100 μg/mouse; anti-CTLA4 Ab, Bio X Cell, 20 μg/mouse); 3# (OVAinMOF)@(anit-CTLA4inMOF) group (OVA, 100 μg/mouse; anti-CTLA4 Ab, 20 μg/mouse; MOF, 600 μg/mouse); and 4# (MS@OVAinMOF)@(anti-CTLA4inMOF) (OVA, 100 μg/mouse; anti-CTLA4 Ab, 20 μg/mouse; MS@MOF, 600 μg/mouse). Each individual subject in 100 μL saline was administered subcutaneously into the left flank of mice at day 0, 3 and 10. At day 14, the mice were challenged by live E.G7-OVA cells (5 × 105 cells mouse−1) subcutaneously into the right flank. Tumour growth on the right flank of mice was monitored three times per week and continued for several weeks. Survival rate was statistically calculated based on tumour size <15 mm. The tumour volume was calculated by 1/2 × longest dimension × perpendicular dimension2.

Combination cancer immunotherapy

First, live E.G7-OVA cells (2 × 105 cells mouse−1) were injected subcutaneously into the right flank of sixty three female C57BL/6 mice (7 mice/group; 6 weeks old, CLEA Inc.). On day 3, 7, 14, 21 post-tumour inoculation, mice were divided into the following nine groups and injected with the following subjects in 100 μL saline: free OVA (100 μg/mouse) plus a high dose of i.p. anti-PD-1 Ab (Bio X Cell, 200 μg/mouse) in group a; only free OVA (100 μg/mouse) in group b; free OVA (100 μg/mouse) plus a low dose of i.p. anti-PD-1 Ab (20 μg/mouse) in group c; OVA absorbed on MS (OVAonMS: OVA, 100 μg/mouse; MS, 600 μg/mouse) plus a low dose of i.p. anti-PD-1 Ab (20 μg/mouse) in group d; OVA/polyIC adsorbed on MS (OVA/polyIConMS: OVA, 100 μg/mouse; polyIC, 100 μg/mouse; MS, 600 μg/mouse) plus a low dose of i.p. anti-PD-1 Ab (20 μg/mouse) in group e; (MS@OVAinMOF)@(polyICinMOF) (OVA, 100 μg/mouse; polyIC, 100 μg/mouse; MS@MOF particles, 600 μg/mouse) plus a low dose of i.p. anti-PD-1 Ab (20 μg/mouse) in group f, OVAonMS (OVA, 100 μg/mouse; MS, 600 μg/mouse) plus a high dose of i.p. anti-PD-1 Ab (200 μg/mouse) in group g; OVA/polyIConMS (OVA, 100 μg/mouse; polyIC, 100 μg/mouse; MS, 600 μg/mouse) plus a high dose of i.p. anti-PD-1 Ab (200 μg/mouse) in group h; (MS@OVAinMOF)@(polyICinMOF) (OVA, 100 μg/mouse; polyIC, 100 μg/mouse; and MS@MOF particles, 600 μg/mouse) into the left flank plus a high dose of i.p. anti-PD-1 Ab (200 μg/mouse) in group i. Tumour growth on the right flank of mice was monitored for several weeks. Survival rate was calculated based on tumour size <15 mm. The tumour volume was calculated by 1/2 × longest dimension × perpendicular dimension2.

Cytokine contents in tumour sites and spleen

At the endpoint of prophylactic and combination cancer immunotherapy, the tumour sites and spleen were excised and lysed with a T-PER tissue protein extraction reagent (Thermo Fisher Scientific), and the amounts of cytokines in tumour sites were quantified using mouse ELISA kit (BD Biosciences) according to the manufacturer’s instructions.

Analysis of antigen-specific T-cell populations

At the endpoint of prophylactic and combination cancer immunotherapy, splenocytes were collected from the spleen, milled, vortexed and filtered through a 40-μm cell strainer to obtain single-cell suspension. Anti-CD16/CD32 antibody (2.4G2, Biolegend) with 1/100 dilution was used to prevent the nonspecific staining. Anti-mouse CD8α Ab (BioLegend) and anti-mouse T-Select H-2Kb OVA Tetramer-SIINFEKL Ab (MBL) with 1/50 dilution were used to stain the cells for 30 min. Then, the intracellular cytokine was stained by anti-mouse IFN-γ Ab (BioLegend) with 1/50 dilution. Flow cytometry was performed for the cell suspensions using a FACSAria cell cytometer (BD Biosciences).

Specific cytotoxic T lymphocyte assay for E.G7-OVA cancer cells

At the endpoint of combination cancer immunotherapy, mice from all groups were killed to harvest splenocytes. After 7 days of splenocytes subculture with 40 ng/mL mouse IL-2 and 20 μg/mL OVA, the splenocytes were cocultured with 5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE, Dojindo) – stained live E.G7-OVA cancer cells and NIH3T3 fibroblasts at effector cells/target cells ratio of 10, respectively. In addition, mouse from group f were cocultured CFSE – stained live E.G7-OVA cancer cells and PC-12 cancer cells at effector cells/target cells ratio of 0, 5, 10 and 20, respectively. The cells were then stained with Ghost Dye™ Violet 450 (Bay bioscience) 24 h later. The cytotoxicity of splenocytes against E.G7-OVA cancer cells, NIH3T3 fibroblasts and PC-12 cancer cells were analysed using a FACSAria cell cytometer (BD Biosciences), respectively. The cytotoxicity is calculated by the following formula: cytotoxicity = (total dead target cells−spontaneous dead target cell)/(total target cells−spontaneous dead target cell) × 100%.

Biocompatibility of nanoadjuvants

To examine the in vivo safety, the saline, MOF-gated MS and MS (1 mg/mouse in 100 μL saline) were subcutaneously injected into the left flank of C57/BL6J mice (5 mice, every group; CLEA Inc.). Mice were euthanized 3 days later, and blood was harvested for blood haematology analysis. The organs, including kidney, spleen, heart, liver and lung, were collected, fixed in 10% neutral buffered formalin solution (Wako), embedded in paraffin and stained with hematoxylin and eosin.

Statistics and reproducibility

The statistical significance of differences was calculated by log-rank test, Student’s t test or ANOVA with Tukey’s multiple comparisons post hoc test. A p value of <0.05 was considered statistically significant. Each experiment was repeated independently at least twice with similar results.

Ethical issue

The animal experiments were permitted by the Ethical Committee of the National Institute of Advanced Industrial Science and Technology (AIST), Japan. All the animal experiments and feeding were carried out in accordance with the guidelines of the Ethical Committee of AIST, Japan.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.



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