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Pharmaceutics 2020, 12, 1102 5 of 17 2.7. Characterization of Various UCNPs The particle size of a series of UCNPs was determined by dynamic light scattering (DLS) (Zetasizer Nano-S90, Malvern Instruments Ltd., Worcestershire, UK) at a 90◦ scattering angle. Irradiation by an 808 nm laser (Laserlab CO, Gyeonggi-do, Korea) was used to identify the fluorescence emission of UCNPs. The morphology of UCNPs was examined using an ultrahigh-resolution analytical electron microscope (HR-TEM) (JEM-3010, JEOL Ltd., Tokyo, Japan). A multimode microplate reader (SynergyTM Neo2, BioTek Instruments, Winooski, VT, USA) was used for the UV-Vis-NIR analysis of the core and shell structures. 2.8. Singlet Oxygen Generation of UCNPs@SiO2-NH2@FA/PEG/TCPP Singlet oxygen sensor green (SOSG) reagents were used to determine the singlet oxygen generation from UCNPs@SiO2-NH2@FA/PEG/TCPP [2,43]. Aliquots (2 mL) of samples of DW, free TCPP, UCNPs@SiO2-NH2@FA/PEG, and UCNPs@SiO2-NH2@FA/PEG/TCPP were mixed with the SOSG reagents (final concentration: 2.5 μM) in a 24-well plate. An 808 nm laser (1.5 W/cm2) irradiated the respective sample wells for DW, UCNPs@SiO2-NH2@FA/PEG, and UCNPs@SiO2-NH2@FA/PEG/TCPP, and a 660 nm laser (10 mW/cm2) was used for free TCPP. While irradiating the laser for 60 min, a 100 μL aliquot of each sample was collected every 10 min, and the fluorescence intensities were determined using a multimode microplate reader (excitation: 485 nm, emission: 538 nm). 2.9. Cytotoxicity Evaluation of UCNPs@SiO2-NH2@FA/PEG/TCPP The cytotoxicity of a series of UCNPs was evaluated by an MTT and a Live/Dead viability assay kit in HeLa cells using a slightly modified previously described procedure [44–46]. For the MTT assay, 100 μL of HeLa cancer cells (1 × 105 cells/mL) was seeded into the wells of a 96-well plate (SPL Life Sciences CO., LTD) and incubated for 24 h. The HeLa cells were treated with each UCNP sample (0, 1, 5, 10, 20, 30 μg/mL as a free TCPP) in DMEM containing 1% FBS and 1% P/S. The concentration of conjugated TCPP onto UCNPs was determined at 660 nm using a UV-Vis spectrometer (VersaMaxTM microplate reader, Molecular Devices, CA, USA) because the TCPP absorbance (660 nm) was not overlapped with those of UCNPs alone and folic acid. After a 4-h incubation, cells were rinsed twice and replenished with fresh DMEM. Hypoxic conditions were obtained by incubating in a hypoxic chamber (5% CO2, 95% N2) for 4 h. Each sample was irradiated with a 660 nm laser (10 mW/cm2) or 808 nm laser (1.5 W/cm2) for 30 min and then incubated for 24 h. After a treatment with 0.5 mg/mL of MTT reagents for 2 h, MTT reagents were removed, and 100 μL of dimethyl sulfoxide (DMSO) was added for 20 min. Finally, the absorbance intensity at 562 nm was analyzed using a UV-Vis spectrometer. Live/DeadTM viability/cytotoxicity kits were used to visualize the death and viability of HeLa cells. For this, 100 μL of HeLa cancer cells (1 × 105 cells/mL) was seeded in a 12-well plate (Ibidi, Lochhamer, Germany) and further incubated for 24 h. The subsequent steps were performed according to the same protocol as for the MTT assay. At 24 h after laser irradiation, cells were washed with PBS twice and stained with Live/Dead assay solution (4 mL of PBS, 8 μL of ethidium homodimer-1, 2 μL of calcein-AM) for 30 min. Stained cells were observed using a confocal laser scanning microscope (CLSM) (LSM510, Carl Zeiss Meditec AG, Jena, Germany). 2.10. Cellular Uptake of UCNPs in HeLa Cells and Spheroids To investigate the cellular uptake of UCNPs@SiO2-NH2@FA/PEG/TCPP, 150 μL of HeLa cells (1 × 105 cells/mL) was seeded in an 8-well plate (Ibidi, Lochhamer, Germany) and incubated for 24 h [46]. HeLa cells were treated with each UCNP sample (20 μg/mL as a free TCPP). At predetermined time points (10, 20, 30, and 60 min), cells were washed three times with PBS and stained with LysoTrackerTM Green and DAPI [47,48]. The cells were observed using a CLSM (excitation of TCPP: 633 nm, emission of TCPP: 650–680 nm) [49]. Separately, the internalization of UCNPs@SiO2-NH2@FA/PEG/TCPP was evaluated in three-dimensional HeLa cell spheroids [2,45,46].PDF Image | Red LED Erbium Upconverting Nanoparticles v Cervical Cancer
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