Recombinant ferritin-based nanoparticles as neoantigen carriers considerably inhibit tumor development and metastasis | Journal of Nanobiotechnology

Mice

Feminine wild-type (WT) C57BL/6 and BALB/c mice have been bought from Beijing Very important River Laboratory Animal Know-how Co., Ltd. (Beijing, China), and feminine OT-1 transgenic mice have been bought from The Jackson Laboratory. All mice used on this examine have been between 6 and eight weeks previous and have been maintained below particular pathogen-free circumstances within the animal facility at Nankai College. All mice have been housed in teams of 5 below circumstances of a 12-hour light-dark cycle (8:00–20:00, gentle; 20:00–8:00, darkish), fixed room temperature (21 °C), appropriate humidity (40%~60%), and free entry to meals and water. The animal procedures have been carried out with moral compliance and approval from the Institutional Animal Care and Use Committee at Nankai College.

Cell traces

The E.G7-OVA, MC-38-OVA, B16F10, 3T3, and LO2 cell traces have been bought from American Kind Tradition Assortment (ATCC, Manassas, VA, USA). The B16F10-OVA cells have been a present from Prof. Music Zhang’s lab. The E.G7-OVA, MC-38-OVA, B16F10-OVA, and B16F10 cells have been grown in RPMI 1640 (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS, Organic Industries) and 1% penicillin-streptomycin (NCM Biotech, Cat. #C100C5). As well as, 50 µM 2-mercaptoethanol (Aladdin, Cat. #M301574) and 0.4 mg/mL G-418 (Solarbio, Cat. #IG0010) have been used for the tradition of E.G7-OVA cells, and 1.5 µg/mL puromycin (Solarbio, Cat. # P8230) was used for the tradition of MC-38-OVA cells to exclude cells not overexpressing OVA. The 3T3 and LO2 cells have been grown in DMEM (Gibco, CA, USA) supplemented with 10% FBS and 1% penicillin-streptomycin. All cells examined unfavourable for mycoplasma contamination in accordance with the ATCC Common Mycoplasma Detection Package (ATCC, Cat. #30–1012 Okay) and have been cultured at 37 °C in a humidified incubator with 5% CO₂.

Protein biosynthesis and purification

The gene encoding Helicobacter pylori ferritin (residues 5-167) was codon-optimized to adapt to the Escherichia coli (E. coli) expression system, and a degree mutation (Asn19Gly) was designed to take away a possible N-linked glycosylation website. The OVA_T peptide (SIINFEKL) adopted by a (GGS)₃ linker and the M30 peptide (PSKPSFQEFVDWENVSPELNSTDQPFL) adopted by a SASGG linker have been fused to the N-terminus of ferritin (residues 5-167) to generate OVA_T-FNs and M30-FNs, respectively [7]. Furthermore, FNs with out peptides on the N-terminus of ferritin (residues 5-167) have been ready as a management. The constructs have been cloned and inserted into the pET-28a (+) expression vector (Novagen, Madison, WI, USA) utilizing the NcoI and XhoI restriction websites. The recombinant plasmids have been remodeled into E. coli BL21(DE3) competent cells (ZOMANBIO, Cat. #ZK201), and the bacterial cells have been grown in LB media supplemented with 50 µg/mL kanamycin (Solarbio, Cat. #K8020) at 37 °C till an absorbance of 0.6 was reached at 600 nm. The proteins have been induced to be overexpressed by 0.7 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Solarbio, Cat. #II0130) for 16 h at 16 °C.

The bacterial cells have been harvested and resuspended in lysis buffer (50 mM Tris, 500 mM NaCl, 5% glycerol, pH 8.0) after which homogenized at a stress of 700 bar. The supernatant containing the recombinant proteins was obtained by centrifugation at 18,000 rpm for 40 min at 4 °C, and the proteins have been remoted by Ni-nitrilotriacetic acid affinity chromatography. Briefly, 2 mL of Ni-NTA resin (TransGen, Cat. #DP101) was used to purify proteins, which have been then equilibrated with PBS (pH 7.2 ~ 7.4) and incubated with the supernatant for two instances. Nontarget proteins have been eliminated by washing with 10 mM, 50 mM, or 100 mM imidazole, and goal proteins have been eluted with 300 mM imidazole. The scale and purity of the harvested proteins have been decided by SDS-PAGE. The eluted proteins have been buffer exchanged into PBS (pH 7.2 ~ 7.4) containing 1 mM EDTA (Solarbio, Cat. #E1170) and 5% glycerol and concentrated to lower than 5 mL utilizing Amicon-Ultra15 centrifugal filters (EMD Millipore) with a 100-kDa molecular weight cutoff (MWCO). The concentrated proteins have been additional purified by measurement exclusion chromatography utilizing a HiPrep 16/60 Sephacryl S-500 h size-exclusion column within the above buffer at a circulation fee of 0.8 mL/min. The purified proteins have been concentrated to 2.5 mg/mL utilizing Amicon-Ultra15 centrifugal filters with a 100-kDa MWCO after elimination of endotoxin in accordance with the producer’s protocols (Thermo, Cat. #88272), after which 5% glycerol was added, adopted by storage at -80 °C.

Destructive-stain electron microscopy

For the negative-staining examine, 4 µL of 100 µg/mL protein pattern was utilized to a carbon film-coated 300-mesh Cu grid (Beijing Zhongjingkeyi Know-how Co., Ltd., Beijing, China) for 1 min. After air-drying, the pattern was negatively stained with 1% (w/v) uranyl acetate for 1 min after which noticed by transmission electron microscopy (TEM) (QUANTA 200, FEI firm, USA) working at 100.0 KV.

Dynamic gentle scattering (DLS) and zeta potential evaluation

Particle measurement and zeta potential evaluation of FNs and OVA_T-FNs at a focus of roughly 2 mg/mL have been carried out utilizing a Zetasizer Nano ZS (Malvern Devices Ltd., UK).

Blood and tissue processing

Roughly 150 µL of peripheral blood was collected right into a 1.5 mL EP tube containing 30 µL of 0.5 M EDTA, and 1.5 mL of ACK buffer (Solarbio, Cat. #R1010) was added to lyse purple blood cells twice for five min every. Lymph nodes or spleen positioned between two 40 μm filters have been mechanically ready into single cells in a 12-well plate containing 1 mL of MACS buffer (1×PBS containing 0.5% BSA and a couple of mM EDTA). As well as, the splenocytes have been lysed at room temperature for five min with 2 mL of ACK buffer to take away purple blood cells. The obtained single-cell suspension was filtered via a 40 μm nylon mesh filter for subsequent circulation cytometry detection or switch experiments.

Preparation of bone marrow-derived dendritic cells (BMDCs)

Bone marrow was flushed from the femurs and tibias of C57BL/6 mice with ice-cold RPMI 1640 medium containing 2% penicillin-streptomycin. Pink blood cells have been lysed, and the remaining cells have been seeded in a ten cm tissue tradition dish containing 12 mL of BMDC medium consisting of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS), 1% penicillin-streptomycin, 50 µM 2-mercaptoethanol, 20 ng/mL IL-4 (PeproTech, Cat. #214 − 14) and 40 ng/mL GM-CSF (PeproTech, Cat. #315-03). On day 2, nonadherent and loosely adherent cells have been collected and transferred to a brand new tradition dish, after which 4 mL of contemporary BMDC medium was added. On day 4, half of the medium was gently eliminated, and an equal quantity of contemporary medium was added. On day 6, BMDCs have been recognized by circulation cytometry and used for subsequent experiments.

In vitro mobile uptake assay

First, appropriate cells cowl glasses (NEST, Cat. #801010) have been positioned in a 24-well plate and 5 × 10⁵ immature BMDCs obtained as described above have been added and cultured for twenty-four h. BMDCs have been then incubated with 2 nmol Cy5-labeled OVA_T, FNs or OVA_T-FNs for 4 h. After the unphagocytosed vaccines have been washed away with PBS, the BMDCs have been stained with CD11c-FITC antibody at 4 °C for 30 min, adopted by fixation with 200 µL of 1% paraformaldehyde (Solarbio, Cat. #P1111) at 4 °C for 20 min. The quilt glasses have been harvested and stained with DAPI, after which the localization of Cy5-labeled antigen in FITC-positive cells was detected by confocal laser scanning microscopy (CLSM; TCS SP5, Leica, Germany).

Analysis of endosomal escape skill

A complete of 1 × 10⁶ immature BMDCs have been grown in 35 mm confocal dishes (NEST, Cat. #801001) for twenty-four h. Then, 2 nmol of FITC-labeled OVA_T, FNs or OVA_T-FNs have been added and cocultured for 4 h. After being washed 5 instances with PBS, the BMDCs have been stained with LysoTracker (Invitrogen, Waltham, MA, USA, Cat. #L7528) for 1 h, adopted by Hoechst (US Everbright, Cat. #H4078) for 30 min. The colocalization of FITC and LysoTracker was detected by confocal laser scanning microscopy (CLSM; TCS SP5, Leica, Germany) to research the endosomal escape skill of FNs and OVA_T-FNs.

Activation and maturation of BMDCs and cross-presentation of OVAT peptide

The cells and tradition supernatant have been harvested after 1 × 10⁶ BMDCs have been cocultured with 2 nmol of OVA_T, FNs or OVA_T-FNs for twenty-four h. BMDCs have been then stained with CD80, CD86, MHC-I, MHC-II, CD40, and SIINFEKL-H2Kb antibodies for 30 min at 4 °C, and the frequencies of activation markers have been analyzed with a BD FACSCalibur Movement Cytometer (BD Biosciences, San Jose, CA, USA). The concentrations of IL-12 (P70), IFN-α1, IL-6, IFN-γ and TNF-α within the tradition supernatant have been decided by enzyme-linked immunosorbent assay (ELISA) in accordance with the producer’s protocols.

In vivo imaging and lymph node focusing on

BALB/c or C57BL/6 mice have been immunized with PBS, 6 nmol Cy5-labeled OVA_T, FNs, or OVA_T-FNs within the groin. The fluorescence intensities on the injection website, remoted lymph nodes and varied organs have been analyzed 24, 48, and 72 h after immunization utilizing an IVIS Lumina imaging system (IVIS Lumina II, Xenogen, USA).

Antigen uptake and activation of APCs in lymph nodes

C57BL/6 mice have been immunized within the groin with Cy5-labeled OVA_T, FNs or OVA_T-FNs at a dose of 6 nmol per facet. Twenty-four hours after immunization, the lymph nodes have been harvested and ready into single-cell suspensions. Then, the frequencies of Cy5 in APCs, corresponding to DCs (CD11c⁺), macrophages (CD11b⁺ F4/80⁺), and B cells (B220⁺), in addition to the frequency of activated DCs (CD80⁺ CD86⁺ CD11c⁺), have been analyzed by circulation cytometry.

In vivo proliferation assay

Splenocytes from OT-1 mice have been labeled with 5 µM CFSE (Invitrogen, Cat. #65–0850) for 1 h at 37 °C after which transferred intravenously to C57BL/6 mice, which have been then immunized subcutaneously within the groin with 6 nmol of OVA_T, FNs and OVA_T-FNs within the presence or absence of fifty µg of Poly(I: C) (InvivoGen, Cat. #tlrl-pic-5) 24 h after switch. The attenuation of CFSE fluorescence depth of OVA_T-specific CD8⁺ T cells within the spleen was analyzed by circulation cytometry at 24, 48, and 72 h after immunization. The proliferation index, representing the whole variety of divisions / cells that went into division, was calculated utilizing the Proliferation Device in FlowJo v10.8.1 software program.

In vivo goal cell lysis assay

Splenocytes from C57BL/6 mice have been resuspended in RPMI-1640 medium supplemented with 1% penicillin-streptomycin, and 50 µM 2-mercaptoethanol, and the cell focus was adjusted to 1 × 10⁷ cells/mL [39]. The above cells have been divided into two aliquots, one in all which was labeled with OVA_T peptide at a last focus of 25 µM for two h at 37 °C, whereas the opposite was unlabeled. Splenocytes loaded with OVA_T peptide and unloaded with OVA_T peptide have been labeled with 5 µM and 0.5 µM CFSE, respectively, for 1 h at 37 °C. The cells have been washed twice with PBS to take away unbound CFSE dye and subsequently blended at a ratio of 1:1 after adjusting the cell focus to five × 10⁷ with PBS. Extra C57BL/6 mice with none therapy have been immunized with PBS, OVA_T, OVA_T+Poly(I: C), OVA_T-FNs, or OVA_T-FNs + Poly(I: C). A complete of seven × 10⁶ blended cells have been injected intravenously into mice immunized for 8 days at a dose of 200 µL per mouse. Eighteen hours after injection, single-cell suspensions of the spleens and lymph nodes have been ready, and the frequencies of splenocytes labeled with excessive and low concentrations of CFSE have been analyzed by circulation cytometry. The precise lysis share was calculated in accordance with the printed components [22]:

Immunizations

A complete of 6 nmol of OVA_T, M30, FNs, OVA_T-FNs, or M30-FNs alone, or ready by mixing with 50 µg of Poly(I: C) have been formulated in 100 µL of PBS and immunized subcutaneously through the groin.

Enzyme-linked immunospot (ELISpot) assay

ELISpot plates (EMD Millipore, MSIPS4510) have been pretreated with 50 µL of 35% ethanol for 30 s and washed 5 instances with sterile water earlier than being coated with 100 µL of 15 µg/mL anti-IFN-γ (MABTECH, Cat. #3321–2 A) in a single day at 4 °C. After coating, the antibody was discarded, and the plate was blocked for 1 h with full medium (RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin). A complete of two × 10⁵ splenocytes have been added to every effectively and restimulated for 48 h at 37 °C with full medium containing OVA_T or M30 peptide at a last focus of 10 µg/mL. The cells have been washed away, and the plates have been incubated with 100 µL of 1 µg/mL biotinylated detection antibody (MABTECH, Cat. #3321–2 A) for two h at room temperature. The plate was washed 5 instances with PBS and incubated with streptavidin-ALP (MABTECH, Cat. #3321–2 A) diluted at a ratio of 1:1000 for 1 h at room temperature. After washing with PBS 5 instances, 100 µL of the substrate NBT&BCIP (Sangon Biotech, Cat. #C510032) ready with 1× colour growth buffer was added to every effectively till apparent spots appeared, after which the response was stopped with 200 µL of ddH₂O. After dying, the spots have been counted with an ELISpot reader (AID iSpot, AID-Autoimmune Diagnostika GmbH, Strassberg, Germany).

Experiments to detect immune responses induced by vaccines

C57BL/6 mice have been immunized on days 0 and 14, and the peripheral blood and spleen have been collected on day 21 to research vaccine-induced immune responses. The frequencies of OVA_T-specific CD8⁺ T cells within the peripheral blood and spleen have been measured by circulation cytometry. The phenotypes of CD8⁺ T cells in peripheral blood have been evaluated by circulation cytometry evaluation of the proportions of PD-1⁺ TIM-3⁻ cells, effector reminiscence T (Tem) cells, and central reminiscence T (Tcm) cells. Vaccine-induced CTL responses have been measured by the secretion of IFN-γ, TNF-α, or Granzyme B by CD8⁺ T cells.

Prophylactic, therapeutic and metastasis mannequin experiments

For prophylactic mannequin experiments, C57BL/6 mice have been immunized on days 0, 14 and 28. Fourteen days after the third immunization, 5 × 10⁵ E.G7-OVA, 1 × 10⁶ MC-38-OVA, or 3 × 10⁵ B16F10-OVA cells in 100 µL of PBS have been subcutaneously implanted into the precise flank of every mouse.

For metastasis mannequin experiments, C57BL/6 mice have been immunized on days 0, 14 and 28, 1 × 10⁵ B16F10 cells in 200 µl of PBS have been injected via the tail vein on day 35, and the lungs of the mice have been eliminated on day 57 to rely the variety of metastatic foci.

For therapeutic mannequin experiments, C57BL/6 mice have been subcutaneously implanted in the precise flank with 1 × 10⁵ B16F10 cells in 100 µL of PBS on day 0 after which immunized thrice on days 5, 8, and 12.

The tumor quantity was estimated by the next components: tumor quantity = size × width² × 0.5. Animals have been euthanized when the tumor reached 1.5 cm in diameter or surpassed 1500 mm³ in quantity or when important weight reduction was noticed.

ELISA

The tradition medium supernatant of BMDCs cocultured with vaccines and of splenocytes restimulated with OVA_T peptide have been collected, and the concentrations of cytokines have been detected by ELISA kits (BioLegend, San Diego, CA, USA). The concentrations of IL-12(p70) (Cat. #433604), IFN-α1 (Cat. #447904), TNF-α (Cat. #430904), IFN-γ (Cat. #430804), and IL-6 (Cat. #431304) have been detected in accordance with the producer’s protocols.

Movement cytometry evaluation

For the APCs uptake evaluation, cells have been preincubated with 0.25 µg of TruStain FcX™ PLUS anti-mouse CD16/32 blocking antibody (BioLegend, clone. S17011E) per 10⁶ cells in a quantity of 100 µl for 10 min on ice to cut back nonspecific binding. After FcR blocking, the cells have been then stained for 30 min at 4 °C with floor antibodies diluted at 1:300 in 100 µL of FACS buffer (fluorochrome-conjugated antibodies bought from BioLegend until in any other case indicated): CD45 (Cat. #103133. clone. 30-F11), CD11b (Cat. #101292. clone. M1/70), CD11c (Cat. #117306 and 117307. clone. N418), CD11c (BD Biosciences, Cat. #612797, clone. HL3), CD80 (Cat. #104705. clone. 16-10A1), CD86 (Cat. #105025. clone. GL-1), CD40 (Cat. #157506. clone. FGK45), MHC-I (Cat. #114612. clone. 28-8-6), MHC-II (Cat. #107625. clone. M5/114.15.2), B220 (Cat. #103205. clone. RA3-6B2), F4/80 (Cat. #123110. clone. BM8), and SIINFEKL-H2Kb (Cat. #141605. clone. 25-D1.16). After staining, the cells have been washed as soon as with FACS buffer and resuspended in 300 µL of PBS for circulation cytometry detection.

For T-cell tetramer evaluation, single cells derived from the peripheral blood or spleen have been first incubated with 100 µL of PBS containing 50 nM dasatinib (MACKLIN, Cat. #D828602) for 30 min at room temperature. The samples have been washed as soon as and handled with an anti-CD16/32 antibody for 10 min on ice. The cells have been then stained with tetramer antibody (MBL, Cat. #TS-5001–1 C) diluted 1:20 in 50 µL of FACS buffer containing 50 nM dasatinib for 45 min at 4 °C below gentle safety. After washing as soon as with FACS buffer, the cells have been stained with floor antibodies diluted at 1:300 in 100 µL of FACS buffer (fluorochrome-conjugated antibodies bought from BioLegend until in any other case indicated) for 30 min at 4 °C: CD8 (GeneTex, Cat. #GTX76348. clone. KT15), PD-1 (Cat. #135209. clone. 29 F.1A12), TIM-3 (Cat. #119718. clone. RMT3-23), CD44 (Cat. #103043. clone. IM7), and CD62L (BD Biosciences, Cat. #564109, clone. MEL-14).

For intracellular cytokine evaluation, 1 × 10⁶ splenocytes have been first stimulated with 10 µg/mL OVA_T peptide and Golgi plug (BD Biosciences, Cat. #555029) at 37 °C for six h. The cells have been then handled with an anti-CD16/32 blocking antibody at 4 °C for 10 min and subsequently stained with CD3 (BD Biosciences, Cat. #564379, clone. 145-2C11), CD4 (Cat. #100491. clone. GK1.5) and CD8 (Cat. #100706. clone. 53 − 6.7) diluted at 1:300 for 30 min at 4 °C. The cells have been washed as soon as with FACS buffer and glued and permeabilized at 4 °C for 1 h utilizing the FoxP3/Transcription Issue Staining Buffer Set (Invitrogen, Cat. #00-5523-00). The cells have been washed as soon as with 1× wash buffer after which stained with the next intracellular antibodies: IFN-γ (Cat. #505808. clone. XMG1.2), TNF-α (Cat. #506313. clone. MP6-XT22), and Granzyme B (Cat. #372216. clone. QA16A02) diluted at 1:300 in 50 µL PBS at 4 °C in a single day. The cells have been washed twice with PBS and ready for circulation cytometry detection.

The cells have been acquired on a BD FACSCalibur Movement Cytometer (BD Biosciences, San Jose, CA, USA) or a BD LSRFortessa X-20 (BD Biosciences) utilizing BD FACSDiva Software program v8.0.3 (BD Biosciences). All collected information have been analyzed with FlowJo model V10.8.1.

CCK-8 assay

The 3T3 cells (5 × 10³), LO2 cells (5 × 10³), and PBMCs (1 × 10⁴) have been seeded into 96-well plates and cultured for twenty-four h. Then, the cells have been handled with completely different doses of FNs and OVA_T-FNs for added 24 h. After therapy, CCK-8 resolution (NCM Biotech, Cat. #C6005) was added to every effectively and incubated for two.5 h. The absorbance was then measured at 450 nm and 600 nm utilizing a Cell Imaging Multi-Mode Reader (Cytation 5, BioTek, Winooski, VT, USA).

Hematoxylin and eosin (H&E) staining and willpower of assorted enzymes in serum

Fourteen days after the third immunization, 200 µL of peripheral blood was collected and positioned at 4 °C in a single day, adopted by centrifugation at 1,000 rpm for 20 min to reap the serum. The center, liver, spleen, lung and kidney have been eliminated and glued in 15 mL of 4% paraformaldehyde (Solarbio, Cat. #P1110) for 4 days. Organ harm and the serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (CRE) have been decided by Tianjin JingNuo Pathological Diagnostic Co.

Statistical analyses

Statistical analyses have been carried out utilizing GraphPad Prism 8.0.2 software program. All outcomes have been offered because the imply ± s.e.m. An unpaired Pupil’s t check, one-way ANOVA with Tukey’s multiple-comparison check, two-way ANOVA with Tukey’s multiple-comparison check or log rank check was used for comparisons between the teams. P values lower than 0.05 have been thought-about to point statistical significance. No pattern in any consultant experiment was excluded from the evaluation.