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Pharmaceutics 2020, 12, 1102 10 of 17 properties, TCPP presents an excellent photo-cytotoxicity for treating and visualizing tumors [68] and absorbs ~660–670 nm light emitted from erbium/lutetium-based UCNPs. Singlet oxygen sensor green (SOSG) is a fluorescent reagent that is highly selective with singlet oxygen and not with hydroxyl radical or superoxide [2,43]. The generated 1O2 causes SOSG to emit a high green fluorescence (maximum excitation/emission wavelengths = ~504/525 nm) by stopping the quenching process of internal electron transfer. In our study, singlet oxygen generation by respective samples was examined for 60 min. In non-TCPP samples (DW and UCNPs@SiO2-NH2 @FA/PEG), the SOSG fluorescence level was negligible at a very low value over 60 min (Figure 5D). On the contrary, UCNPs@SiO2-NH2@FA/PEG/TCPP had almost the same singlet oxygen-generating activity as free TCPP at normalized concentrations of TCPP, and the SOSG fluorescence gradually increased in proportion to the laser irradiation time (Figure 5D). This result indicated that UCNPs@SiO2-NH2@FA/PEG/TCPP efficiently absorbed 808 nm light and emitted ~660 nm light that activated TCPP to form singlet oxygen. Once again, the efficiency of singlet oxygen generation appeared to be greatly reinforced by the unique ability of the 660 nm-emitting UCNPs platform. 3.5. Cytotoxicity Assay for Various UCNPs in Normoxic and Hypoxic Conditions The cytotoxicities of various UCNPs, including UCNPs@SiO2-NH2@FA/PEG/TCPP, toward HeLa cells were evaluated through an MTT assay under normoxic and hypoxic conditions. HeLa cells were treated with different concentrations (0, 1, 5, 10, 20, 30 μg/mL) of TCPP-containing samples that were previously normalized as free TCPP, and received 660 and 808 nm laser irradiation for free TCPP and UCNPs, respectively. As shown in Figure 6, in the absence of laser irradiation, all experimental groups showed a low cytotoxicity regardless of the TCPP concentration and oxygen presence. UCNPs@SiO2-NH2@FA/PEG (without TCPP) displayed an insignificant cytotoxicity either with or without 808 nm light regardless of oxygen due to no lack of singlet oxygen generation. However, UCNPs@SiO2-NH2@FA/PEG/TCPP appeared to generate singlet oxygen due to a clear cytotoxicity. In fact, UCNPs@SiO2-NH2@FA/PEG/TCPP killed ~80% of HeLa cells at 30 μg/mL under 808 nm laser irradiation. The overall cytotoxicity degree of UCNPs@SiO2-NH2@FA/PEG/TCPP plus the 808 nm laser was almost the same as that of free TCPP plus the 660 nm laser, which indicated the effective upconversion of our sample for tumor ablation. Nevertheless, the cytotoxicity of UCNPs@SiO2-NH2@FA/PEG/TCPP (70% cell viability) was significantly reduced under hypoxic conditions. 3.6. Live/Dead Assay for Various UCNPs in Normoxic and Hypoxic Conditions In live/dead assays, both seeded 2D monolayers and 3D spheroids of HCT 116 cells were treated with PBS, free TCPP, and UCNPs@SiO2-NH2@FA/PEG/TCPP with or without 808 nm laser irradiation. The overall cytotoxicity results from the Live/DeadTM assay were similar to those of the MTT assay. CLSM images showed the degree of cell death (green emission: live cell, calcein-AM/red emission: dead cell, ethidium homodimer-1). When only PBS was applied, a strong green emission was seen in all samples in normoxic and hypoxic conditions regardless of laser irradiation (Figure 7). In free TCPP, however, almost all cells were killed under a 660 nm laser or normoxic conditions, whereas most cells were live under a no-laser or hypoxic conditions, showing a low intensity of red fluorescence. As with the MTT assay, UCNPs@SiO2-NH2@FA/PEG had a weak cell killing effect in all cases of laser or oxygen presence. In contrast, UCNPs@SiO2-NH2@FA/PEG/TCPP showed a strong red emission upon 808 nm laser irradiation, and all HeLa cells appeared to be dead in normoxia. Laser irradiation did not show a significant effect of cancer cell death due to a low production of singlet oxygen in hypoxic conditions. Nonetheless, a significant fraction of cells was dead even in hypoxia for UCNPs@SiO2-NH2@FA/PEG/TCPP plus the 808 nm laser. As shown in Figure 8A,B, UCNPs@SiO2-NH2@FA/PEG/TCPP displayed a clear cytotoxicity to cells of ~300 μm-spheroids in normoxia with the 808 nm laser, as proven by the widespread red fluorescence color, and penetrated deep into the spheroids (10–80 μm Z-track images from slices with a 10-μm step size). However, a negligiblePDF Image | Red LED Erbium Upconverting Nanoparticles v Cervical Cancer
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