Synthesis of CeO2 nanoparticles
CeO2 nanoparticles have been synthesized by thermal decomposition of Cerium (III) nitrate hexahydrate (Ce(NO3)3·6H2O) [15]. Initially, 3 mmol of cerium (III) nitrate hexahydrate (99%, Sigma-Aldrich) have been dissolved in 10 ml of 1-octadecene at room temperature. Subsequently, 1 mmol of oleylamine was added to the combination, which was then heated to 120 °C below an argon environment and stirred for 1.5 h. This was adopted by further heating to 270 °C for 1 h and, lastly, to 300 °C for 20 min. The nanoparticles have been precipitated utilizing ethanol, resuspended in tetrahydrofuran (THF), and saved at 4 °C for subsequent evaluation.
Preparation of OMVs and CeO2@OMVs
For preparation of OMVs, E. coli Competent Cells (ECOSTM BL21 (DE3), Yeastern Biotech Co., Ltd., Taiwan) have been cultured within the Luria Broth (LB) medium for 18–24 h at 37 °C in a shaking incubator (250 rpm) till the OD600 reached 0.8 [16]. After centrifuging at 5,000 rpm for two h, the cell pellet was resuspended in Tris/EDTA/NaCl buffer and shaken at 57 °C for 1 h. Membrane disruption was achieved utilizing a Excessive-Stress Cell Disruptor (FRENCH Press TS collection, Fixed Techniques TS-0.75 kW) below 33 psi, adopted by centrifugation at 13,200 rpm for 30 min. The supernatant was collected and centrifuged at 14,000 rpm for 1 h, adopted by a second centrifugation at 36,500 rpm for two h at 4 °C utilizing a SW 41 Ti rotor (Beckman Coulter Inc., Brea, CA, USA). These unpurified OMVs have been then added to the density gradient established by totally different concentrations of cesium chloride (CsCl) options (1.1 g/ml, 1.2 g/ml, and 1.3 g/ml). Following centrifugation at 36,500 rpm for 4.5 h at 4 °C, the OMVs have been collected from the layer with a density of 1.2 g/ml CsCl. After a ultimate centrifugation at 36,500 rpm for two h at 4 °C, the harvested OMVs have been resuspended in phosphate buffered saline (PBS) and saved at -20 °C.
For synthesis of CeO2@OMVs, the OMVs have been blended with CeO2 with a ratio of 10:1 in a high-pressure homogenizer (Nanolyzer N2, Gogene Company, Hsinchu County, Taiwan). The OMVs have been mechanically disrupted below excessive strain (10,000 psi), inflicting transient disintegration of the bilayer membrane construction. Concurrently, a CeO2 nanoparticle suspension (in Hexane/THF cosolvents) was step by step launched into the combination. Upon completion of the strain cycles, the OMVs self-assembled, embedding CeO2 nanoparticles inside the hydrophobic core of the reassembled bilayer membrane by way of hydrophobic interactions. Extra cycles at a decrease strain (5,000 psi) have been carried out to make sure uniform particle dimension and stop aggregation. The ensuing CeO2@OMVs have been precipitated by centrifugation at 13,200 rpm for 20 min after which resuspended in PBS, with a yield proportion of 11.9%.
Characterization of CeO2@OMV
The morphologies of CeO2, OMVs, and CeO2@OMVs have been examined utilizing transmission electron microscopy (TEM; JEOL, JEM-2000 EX II, Japan). The particle dimension distribution and zeta potential of the samples was decided utilizing dynamic gentle scattering (DLS; Zetasizer Nano ZS90, Malvern Devices, Worcestershire, UK) at a scattering angle of 90° at 25 °C. The protein elements of the samples have been decided utilizing a Bradford protein assay [17].
Cell traces
Murine breast most cancers cell line 4T1 (ATCC® CRL-2539™) was obtained from American Sort Tradition Assortment (ATCC). Murine macrophages RAW 264.7 (BCRC 60001) have been obtained from the Bioresource Assortment and Analysis Middle (BCRC). These cells have been cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone) containing 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin-streptomycin. L929 (BCRC 60091) murine fibroblasts, acquired from BCRC, have been cultured in Minimal Important Medium with Earle’s Balanced Salts (MEM/EBSS, HyClone) supplemented with 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10% FBS and 1% penicillin-streptomycin. Cells have been maintained in a 37 °C incubator with humidified air supplemented with 5% CO2. The 4T1-Luc cells (luciferase-expressing cells from ATCC® CRL-2539) engineered by Academia Sinica, have been cultured with 4 µg/ml puromycin for luciferase-expressing cell choice.
Cell viability assay
The cell viability was evaluated utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [18]. The 4T1 and L929 cells have been seeded at a density of 6 × 104 cells per nicely in a 24-well plate. The cells have been co-cultured with totally different concentrations (50, 25, 12.5, 6.25, 3.125 µg/ml) of OMVs and CeO2@OMVs for twenty-four h. Following PBS washing, 5% PrestoBlue™ reagent (Thermo Fisher Scientific, Canada) was added and incubated for 30 min to judge the cell viability. The supernatant was then transferred to a 96-well plate at 100 µl/nicely and the absorbance was measured utilizing a Tecan Dawn ELISA Reader (Mannedorf, Zurich, Switzerland) with excitation/emission set at 560/590 nm.
Intracellular accumulation
To evaluate intracellular uptake, 4T1 cells (5 × 104 cells/nicely) handled with PBS, OMVs or CeO2@OMVs for twenty-four h have been fastened with 4% paraformaldehyde after which incubated in 0.1% Triton X-100 (Sigma) for 10 min. F-actin and cell nuclei have been stained with Alexa Fluor™ 488 Phalloidin (Invitrogen) and Fluoroshield with DAPI (GeneTex), respectively. The intracellular uptake of CeO2@OMVs was examined by confocal microscopy (Zeiss LSM880), revealing pink fluorescence upon excitation at 543 nm.
Intracellular ROS detection
The intracellular ROS manufacturing was decided utilizing move cytometry. 4T1 cells (8 × 105 cells/nicely) have been seeded into 6-well plates and handled with PBS, OMVs or CeO2@OMVs for twenty-four h, adopted with or and not using a single fraction of 4 Gy (Gy) X-ray irradiation (XR, i.e., 6-MV photons at a dose charge of 100 rad/min). At 30-minute post-irradiation, the cells have been labeled with CellROX™ Deep Purple Reagent (5 µM) and the ROS ranges have been quantified by move cytometry (Beckman Coulter CytoFLEX).
Gamma (γ)-H2AX assays
The DNA injury was decided by the Gamma-H2AX assay [19]. 4T1 cells (5 × 104 cells/nicely) have been seeded into 12-well plates. The cells have been handled with PBS, OMVs or CeO2@OMVs for twenty-four h, with or and not using a single fraction of 4 Gy XR. At 30-minute post-irradiation, the handled cells have been fastened utilizing paraformaldehyde (4%) and incubated in 0.1% Triton X-100 (Sigma) for 10 min. The cells have been incubated with anti-gamma H2AX (ab22551, 10 µg/ml) in a single day at 4°C. Following day, the secondary antibodies, anti-mouse IgG (DyLight R 488), have been added and incubated for 1 h at room temperature. Nuclei have been mounted with Fluoroshield with DAPI (GeneTex). The fluorescent photographs have been noticed utilizing an upright fluorescent microscope (Leica DM6000B, Switzerland).
Transwell co-culture with macrophages
Within the Transwell® cell tradition inserts with 0.4 μm pored-filters, 3 × 105 4T1 cells have been seeded on the higher inserts and co-cultured with RAW 264.7 macrophages positioned within the decrease chamber. The 4T1 cells have been handled with PBS, OMVs or CeO2@OMVs for twenty-four h, with or with out XR with 4 Gy per fraction for 2 consecutive days. The conditioned medium was collected and utilized to an impartial tradition of unirradiated 4T1 cells for added 24 h. For cell proliferation assay, cells have been labeled with 5% PrestoBlue™ reagent (Thermo Fisher Scientific, Canada) and the absorbance was measured utilizing a Tecan Dawn ELISA Reader (Mannedorf, Zurich, Switzerland) with excitation/emission set at 560/590 nm. The bystander impact on cell viability was additional assessed by a Calcein-AM/PI twin staining assay [20]. Cells have been incubated with Calcein-AM (2 µM) and Propidium Iodide (PI, 4 µM) in serum-free medium for 30 min at 37 °C. Reside cells and lifeless cells have been visualized utilizing a fluorescence microscope (Leica DM6000B, Switzerland). For wound therapeutic assay, a scratched space was created within the tradition utilizing a sterile tip. Adjustments within the scratched space have been recorded utilizing an inverted microscope (Nikon, TE2000-U, USA) and quantified utilizing ImageJ software program (Nationwide Institutes of Well being, USA).
Cell migration and invasion assays
Bystander 4T1 cells have been resuspended in varied conditioned media and utilized for cell migration and invasion assays. Within the cell migration assay, 4T1 cell suspension was instantly seeded onto the higher insert of the Transwell® system with 8 μm-pored membranes. Within the cell invasion assay, 100 µl diluted Matrigel was added to the higher insert to kind a gel matrix. The process was just like the migration assay, with the exception that cell suspensions have been seeded onto the Matrigel. For each assays, cells have been fastened and stained with 0.05% crystal violet after 24-hour incubation. The variety of migrated or invaded cells have been noticed utilizing an inverted microscope (Nikon, TE2000-U, USA) and quantified by ImageJ software program (Nationwide Institutes of Well being, USA).
Western blot evaluation
The expression ranges of cell proteins have been quantified by way of western blotting evaluation. Cells have been lysed utilizing RIPA buffer (Biotools) and ultrasonic disruption (Misonix, L2000). Proteins have been separated by way of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated with main antibodies at 4 °C in a single day. Subsequent steps concerned washing with PBS and incubating with secondary antibodies at room temperature for 1 h. The intensities of the bands have been quantified by the Amersham Imager 600/680 system (GE Healthcare Life Sciences, Chicago, IL, USA).
Orthotopic two-tumor mannequin
Feminine BALB/c mice (three-to-four-week-old) have been bought from BioLASCO Taiwan Co., Ltd. All animal experiments have been performed in accordance with Nationwide Yang Ming Chiao Tung College (NYCU)’s Institutional Animal Care and Use Committee (IACUC) insurance policies and permitted by IACUC of NYCU (NYMU-IACUC 1110320rr).
To ascertain a bilateral orthotopic 4T1 tumor-bearing mice, 5 × 105 4T1 cells have been injected into the proper mammary fats pad of the feminine BALB/c mice (main tumors) on day 0, and 5 × 104 4T1 cells into the left mammary fats pad of the identical mouse (distant tumors) on day 4. As soon as profitable tumor induction on either side was confirmed utilizing an in vivo imaging system (IVIS Lumina III imaging system, PerkinElmer, UK) on day 7, mice have been randomly assigned to totally different therapy teams, together with (1) Management (2), OMV (3), CeO2@OMV (4), Management + XR (5), OMV + XR, and (6) CeO2@OMV + XR. The first tumors obtained intratumoral injection of two.5 µg/10 µl of OMVs or CeO2@OMVs and have been irradiated with 4 Gy per fraction on the subsequent three consecutive days as specified for every group. Mice have been anesthetized utilizing intraperitoneal injection earlier than irradiation. To make sure exact irradiation, mice have been positioned below a lead shielding equipment, exposing solely the first tumors to irradiation, and the distant tumors remained unexposed all through the experiment. Adjustments within the size and width of tumors have been measured utilizing a digital caliper, and tumor sizes have been calculated utilizing the method (size × width2) / 2. Every therapy arm consisted of 4 mice, and all experiments have been repeated 3 times.
Histopathological evaluation
All of the 4T1 tumor-bearing mice have been euthanized on day 23. The distant tumors and lung tissues have been harvested and glued. Paraffin-embedded sections have been stained with hematoxylin and eosin (H&E) and examined below a lightweight microscope. The mitotic index was decided by the typical variety of mitotic cells in 10 high-power fields [21]. The share of stromal tumor-infiltrating lymphocytes (TILs) have been quantified following the rules of the Worldwide Immuno-Oncology Biomarker Working Group by skilled veterinary pathologists [22, 23]. Immunohistochemistry (IHC) was carried out on formalin-fixed, paraffin-embedded tissue sections. After deparaffinization, rehydration, and heat-induced antigen retrieval, endogenous peroxidase was blocked with 3% hydrogen peroxide. Sections have been then blocked with 5% BSA, incubated with main antibody in a single day at 4 °C, adopted by a secondary antibody (CD4, #50134-R0001, Interchim; CD8, #AB203035, Abcam). Every tissue part was examined in at the least 6 random fields and analyzed with the ImageJ software program (Nationwide Institutes of Well being, USA). Quantification of the CD4+ and CD8+ T cells was expressed as the typical of 10 high-power fields.
Statistical evaluation
Statistical evaluation was performed utilizing Prism 8.0 (GraphPad Software program, Inc., CA). In vitro experiments have been independently repeated at the least 3 times. The outcomes are expressed as imply ± customary deviation. Steady variables have been in contrast utilizing Pupil’s t-test or Mann-Whitney check. Statistical distinction of tumor progress curves was carried out utilizing Two-way ANOVA check. A p worth < 0.05 was thought of statistically important.
