Home Nanotechnology Mannose-doped metal-organic frameworks induce tumor cell pyroptosis through the PERK pathway | Journal of Nanobiotechnology

Mannose-doped metal-organic frameworks induce tumor cell pyroptosis through the PERK pathway | Journal of Nanobiotechnology

Mannose-doped metal-organic frameworks induce tumor cell pyroptosis through the PERK pathway | Journal of Nanobiotechnology


Chemical compounds and reagents

Ferric chloride hexahydrate (FeCl3·6H2O), 1,3,5-Bfienzenetricarboxylic acid (H3BTC), and C2H5OH have been bought from Aladdin (Shanghai, China). Z-YVAD-FMK was bought from Selleck (Shanghai, China). Liproxstatin-1 was bought from ApexBio (Houston, USA). Mannose and 4-Phenylbutyric acid (4-PBA) have been bought from Sigma (NY, USA). FITC was bought from MCE (NJ, USA). The polyvinylidene difluoride (PVDF) membrane was bought from Merck (Darmstadt, Germany). The BCA protein assay equipment, DCFH-DA, Fluo-4am, LDH Cytotoxicity Assay Package, RIPA lysis buffer, and H&E staining equipment have been bought from Beyotime Biotech (Nantong, China). Antibodies in opposition to GSDMD, N-GSDMD, PERK, ATF4, and cleaved Caspase-1 have been bought from Abcam (Shanghai, China). ASC, IL-1β, CD8, CD4, CD11c, and Foxp3 have been bought from Santa Cruz (SC, USA). CHOP, CD206, and tubulin have been bought from Proteintech (Wuhan, China). EIF2α and p-eIF2α have been obtained from CST (Beverly, MA, USA). The Annexin V/PI double stain equipment and superenhanced chemiluminescence (ECL) detection reagent have been bought from Yeasen (Shanghai, China). TNF-α and IL-1β ELISA kits have been bought from ABclonal (Wuhan, China).

Cell tradition

Human and mouse oral squamous cell carcinoma cells (CAL27/SCC-7) and human immortalized keratinocytes (HaCaT) have been bought from ATCC, as indicated. All cells have been cultured at 37 °C and 5% CO2 in DMEM/1640, supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin.

Preparation of Fe3O4

Fe3O4 nanoparticles have been synthesized utilizing a solvothermal discount technique. Usually, 0.13 g of FeCl3·6H2O in 8 mL ethylene glycol was stirred for 30 min at room temperature. Then 1.3 g of sodium acetate was added to the answer and stirred for one more 1 h. The ensuing resolution was transferred right into a Teflon tube, sealed, and heated for twenty-four h at 180 oC. Lastly, the ensuing magnetic Fe3O4 nanoparticles have been centrifuged and washed with water thrice, then redispersed in 10 mL H2O for additional use.

Preparation of Fe3O4@NH2-MIL-100 (FNM)

Amino-modified H3BTC was synthesized in response to a earlier work [15]. First, 2.7 g of FeCl3·6H2O was dissolved in 1 L C2H5OH, and a pair of.5 g of amino-modified H3BTC (NH2-H3BTC) was additionally dissolved in 1 L C2H5OH. Then, 5 mL of above Fe3O4 suspension and 10 mL of FeCl3 resolution sonicated for 30 min, then centrifuged with out wash. 10 mL of NH2-H3BTC resolution was added and heated for 30 min at 65 oC. Then, the combination was centrifuged and washed with ethanal. Repeat this process for 8 occasions, adopted by air drying for additional use.

Preparation of Mannose-doped Fe3O4@NH2-MIL-100 (M-FNM)

Fifty milligrams mannose was dissolved in 50 mL Phosphate buffered saline. 5 mg Fe3O4@NH2-MIL-100 nanoparticles was added to 10 mL mannose resolution, saved 24 h at room temperature. The ultimate product was collected by centrifugation and washed by PBS, adopted by air drying.

Transmission electron microscopy (TEM) evaluation

The samples have been dispersed on a copper grid coated with a carbon movie. The morphology and construction of the samples have been decided by JEM-2100 TEM (JEOL, Tokyo, Japan) with an accelerated voltage of 200 kV. Analyze photos utilizing the Gatan Microscopy Suite software program (Las Vegas, Nevada, USA).

N2 adsorption evaluation

N2 adsorptions isotherm was measured on Quantachrome Autosorb-IQ2 liquid nitrogen computerized volumetric analyzer Bathtub (77 Okay). Utilizing ultra-high purity N2 for adsorption experiments. This BET floor space evaluation was carried out by plotting x/v (1-x) vs. x, the place x = P/P0 (P0 = 1 bar), the place v is the quantity temperature and stress (STP) of nitrogen adsorbed per gram of M-FNM beneath commonplace situations, with passable correlation coefficients and constructive C constants have been noticed. The slope ([c-1]/vmc) and y intercept (the linear area between the dashed line of 1/vmc) present the single-layer capability vm, which is used to calculate the capability from A = vmσ0NAV, the place σ0 is Avogadro’s cross-sectional space of the adsorbate at liquid density (16.2 Å2 for nitrogen) and NAV.

X-ray diffractometer (XRD) evaluation

The XRD sample was recorded on a powder X-ray diffractometer (XRD, Rigaku D-MAX 2500/PC) geared up with a rotating anode and Cu/Kα1 radiation supply (λ = 1.5406 Å), with a step measurement of 0.01°. Use Mercury software program to simulate the XRD sample of Mannose, Fe3O4, and MIL-100 (Fe) from crystallography information.

Fourier rework infrared (FT-IR) spectroscopy

For FT-IR evaluation, 20 mg KBr was floor along with 0.5 mg of the pattern, after which pressed at 10 MPa. The spectrum was recorded on an FT-IR spectrophotometer (Thermo Scientific Nicolet iN5, Waltham, MA, USA) with a wavelength vary of 400–4000 cm− 1. All collected information was processed utilizing OMNIC Specta (Thermo Scientific, Carlsbad, CA, USA).

X-ray photoelectron spectroscopy (XPS) evaluation

The photoelectron spectrometer makes use of Axis Extremely imaging photoelectrons for evaluation. The spectrometer (Thermo Scientific Okay-Alpha, Waltham, MA, USA) is provided with Al Okay α (1486.7 eV) Quartz monochromator gentle supply. Throughout measurement, all samples have been handed by plastic tape. The carbon C1s sign with binding power of 284.8 eV is corrected by setting graphite as reference. All collected information was processed utilizing Avantage software program.

Power dispersive X-ray (EDX) evaluation

The weather distribution was characterised utilizing a scanning transmission electron microscopy (STEM, JEM-2100 F) with EDX.

Cell viability

Regulate the density of CAL27 cells in logarithmic progress part to 1×105 cells/mL, take a 96-well plate, add 100 µL of cell suspension to every effectively, and place it in an incubator to tradition in a single day. Configuration containing detection M-FNM at completely different concentrations of 10, 20, 40, 80, 100, 200, and 300 µg/mL. After the cells are fully adherent, aspirate the medium and substitute with the ready medium. On the similar time, a medium management group (destructive group) and a clean management group containing solely medium with out cells have been set, with 6 replicate wells in every group. For inhibition experiments by Z-YVAD-FMK and Liproxstatin-1. Z-YVAD-FMK was saved at 2 mM in 10% DMSO resolution. 1 µL was added to 100 µL medium within the 96-well plate. Liproxstatin-1 was saved as 10 mM resolution in DMSO. After 1:10 dilution in EtOH, 1 µL was added to 100 µL medium within the 96-well plate. After 24 h, add 10 µL of CCK-8 reagent to every effectively, proceed to incubate for 1 h, and file the absorbance (OD) worth of the microplate reader at a wavelength of 450 nm.

Measurements for the intracellular ROS and focus of Ca2+

Cells have been planted onto confocal dish and cultured in a single day. After that, cells have been incubated with M-FNM (focus: 300 µg/mL) for 4 h, respectively. The ROS ranges have been measured utilizing the DCFH-DA reagent. The cells have been incubated with DCFH-DA at a ultimate focus of 10 µM in FBS-free DMEM for 30 min at 37 °C at midnight, washed thrice with FBS-free DMEM and resuspended in ice-cold PBS for the observe of ROS utilizing a confocal laser scanning microscopy. The intracellular Ca2+ focus was measured by the Fluo-4 am probe, and washed by PBS twice for confocal microscopy commentary.

Electron spin resonance (ESR) detection

The cells have been inoculated into tradition dishes, and after M-FNM therapy, the cell supernatants from all teams have been collected. Dimethyl-1-pyrroline-N-oxide (DMPO) was chosen because the seize agent for hydroxyl radicals (•OH), and the detection was carried out following the process.

Mobile uptake pathways

To discover the uptake pathway and effectivity of M-FNM by CAL27 cells, the precise experimental steps are as follows: 1×104 CAL27 cells/effectively have been seeded in confocal dishes and incubated for twenty-four h. The experimental teams: management group, FNM group, M-FNM group and Mannan + M-FNM group (mannan pretreated cells for 1 h with the focus of 80 µg/mL). The medium of FITC-labeled FNM/M-FNM (focus of 100 µg/mL) have been additional incubated at midnight for 1 h. Wash the cells thrice with PBS to take away extra FNM/M-FNM. Cells in every group have been fastened with absolute ethanol for 10 min, after which the nuclei have been labeled with DAPI. The fluorescence sign of the cells was noticed with a laser confocal microscope and the fluorescence depth was analyzed with ImageJ.

Detection of M-FNM-induced pyroptosis MCSs

Plate cells into Nunclon Sphera effectively plates at a density of 1×106/effectively; incubate plates at 37 °C and 5% CO2; monitor MCSs formation for as much as 7 days; and the cultures have been modified each 72 h, fastidiously take away 500 µL of medium from every effectively and complement with 500 µL of contemporary medium. The M-FNM (300 µg/mL) have been incubated with MCSs 48 h, then by immunofluorescence staining for cleaved Caspase-1, N-GSDMD and DAPI. Lastly, the fluorescence indicators of the MCSs have been noticed utilizing a confocal microscope.

Western blot evaluation

CAL27/SCC-7 cells (1×105) have been seeded into 6-well plate. 24 h after seeding the cells, M-FNM (300 µg/mL) have been added into 3 wells of the plate. the focus of 4-PBA is 5mM and pretreated cells 1 h. The working focus of nigericin was 10 µg/mL, and the cells have been handled concurrently with the M-FNM group. The opposite wells with none therapy and have been used for the management measurements. On the onset of cell lysis, cells have been washed twice with PBS and frozen at -80 °C. The wells that have been handled equally have been pooled for western blot assay. Western blot was carried out in response to commonplace procedures. The protein quantity was normalized to the complete protein content material through the Bradford assay and the outcome was normalized to the untreated management.

Pyroptosis assays

Take the well-grown cells and unfold them evenly into the tradition plates or dishes wanted for every experiment. The small print of every therapy are as follows: to detect cell morphology/IL-1β/LDH launch, 4-PBA (5 mM) was added 1 h upfront after which M-FNM (300 µg/mL) was added for twenty-four h. The management group acquired no therapy. To look at cell morphology, annexin V/PI have been added to the cell-culture medium earlier than subjected to imaging on a confocal microscope. The TEM is utilized to look at the microscopic structural adjustments of CAL27 cells. The picture information proven are consultant of no less than three randomly chosen fields. For the IL-1β ranges within the supernatant, CAL27 cells in logarithmic progress part have been evenly distributed within the 6-well plate, making certain a cell density of about 1×105/effectively. The cells have been positioned in a cell tradition incubator and incubated for twenty-four h for subsequent assays. Membrane integrity was analyzed by detecting the exercise of LDH launched into cell tradition supernatants utilizing the LDH cytotoxicity assay equipment in response to the producer’s protocol.

Immunofluorescence staining

CAL27 cells have been cultured as described within the western blot experiments. Cultured cells have been initially fastened with 4% PFA (paraformaldehyde), permeabilized with 0.5% Triton X-100 and blocked with 1% BSA/PBST. Main antibodies have been subsequently utilized and incubated at 4 °C for twenty-four h, after which rinsed with PBS thrice. After the cells rinsing with PBS, they have been incubated with secondary antibody at 37 °C for 1 h, rinsed with PBS thrice, after which counterstained with DAPI (10 µg/mL) and imaged utilizing a confocal microscope.

To detect the infiltration of immune cells, immunofluorescence was carried out and tumor histology was assessed in response to the usual steps of tissue processing; then, the sections have been additional processed by immunofluorescence. The pictures have been collected beneath a confocal microscope and the fluorescence depth was analyzed utilizing ImageJ.

The biodistribution of nanoparticles in vivo

The procedures adopted for each day animal care and experiments have been carried out beneath particular pathogen-free (SPF) situations with an ambient temperature of 24 ± 2 °C, an air humidity of 40 − 70%, and a 12 h darkish/12 h gentle cycle. BALB/c-nude mice (male, 5 weeks previous) have been bought from SPF (Beijing) Biotechnology (Beijing, China). BALB/c-nude mice acquired a subcutaneous injection of 1 × 106 CAL27 cells in the suitable flank to ascertain a strong tumor-bearing mouse mannequin. After the tumor quantity reached 200 mm3, the mice have been intravenously administered Cy5.5-labeled FNM or M-FNM. Counting on the fluorescence of Cy5.5, a live-imaging system (PerkinElmer IVIS Spectrum, USA) was utilized to visualise the distribution of various brokers in vivo. Eight hours after intravenous (i.v.) administration, the mice have been sacrificed to gather main organs and tumors for in vivo imaging.

M-FNM inhibited Tumor and activated antitumor immunity

BALB/c-nude mice acquired subcutaneous injection of two × 105 CAL27 cells (a human oral squamous cell carcinoma cell line) on the proper flank. 7 days submit tumor inoculation, mice have been randomly divided into 5 teams (5 mice per group), and acquired tail intravenous injection of PBS, mannose, Fe3O4, FNM, and M-FNM (Dosing frequency: each three days; Concentrations: 10 mg/kg). After that, tumor measurement and mouse weight have been recorded for 12 days after tumor formation. Concurrently, to evaluate the function of M-FNM-mediated anti-tumor immunity, a SCC-7 (a mouse oral squamous cell carcinoma cell line) tumor-bearing C57BL/6J mouse mannequin was constructed and mice acquired the identical remedies. The C57BL/6J mice (male, 5 weeks previous) have been bought from SPF (Beijing) Biotechnology (Beijing, China). The infiltration of immune cells is detected in response to the above immunofluorescence technique. For cytokine evaluation, serum samples have been collected from pretreated mice and diluted earlier than measurement. TNF-α and IL-1β have been detected utilizing ELISA kits in response to the producer’s protocol.

Toxicological evaluation

For the histological evaluation, tumor-bearing C57BL/6J mice have been killed after the indicated remedies, and the center, liver, spleen, lung, and kidney have been eliminated and stuck in a single day with 4% formalin. After dehydration by gradient ethanol therapy, the tissue samples have been embedded in paraffin and sectioned for hematoxylin and eosin staining. To confirm the long-term organic security of M-FNM, we administered a tail vein injection each three days to C57BL/6J mice for a complete of 42 days. The injections included M-FNM and different teams (10 mg/mL). Gathering plasma from all teams for testing indicators equivalent to creatinine (Cre), creatinine kinase (CK), lactic acid dehydrogenase (LDH), and aspartic acid transaminase (AST).

Statistical evaluation

Statistical information have been expressed as means ± commonplace deviation (S.D.). Comparisons between completely different teams have been carried out with Scholar’s t-test and evaluation of variance (ANOVA) as acceptable. Values of P < 0.05 have been thought-about to be statistically important.



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