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Pharmaceutics 2020, 12, x FOR PEER REVIEW 10 of 16 UCNPs@SiO2-NH2@FA/PEG/TCPP had almost the same singlet oxygen-generating activity as free Pharmaceutics 2020, 12, 1102 11 of 17 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- cNyHto2t@oFxAici/tPyEwGa/TsCshPoPwenfficnietnhtelyspahbesororibdesdt8re0a8tnedmwliigthtUaCndNePms@itSteiOd ~-6N6H0 n@mFlAig/hPtEtGh/aTtCacPtPiviantehdyTpCoxPiPa 22 wtoitfhortmhesi8n0g8lentmoxlyasgerna.nOdncineangoarimn,otxhieaewffiitchieonuctythoefs8i0n8gnlemtolxaysgeren(Fgigenuerera8tiBo)n.Tahpipsefianreddintogbinedgirceaatteldy trheaintfUorCceNdPbsy@tShiOe u-nNiqHue@aFbAil/iPtyEoGf/TthCeP6P60wnomu-ledmpirtetisnugmUaCblNyPhsapvleattfhoermp.ractical potential to have a 22 photodynamic antitumor efficacy for the relevant tumor in vivo. 3.5. Cytotoxicity Assay for Various UCNPs in Normoxic and Hypoxic Conditions 3.7. Cellular Uptake of UCNPs@SiO2-NH2@FA/PEG/TCPP into HeLa Cells The cytotoxicities of various UCNPs, including UCNPs@SiO2-NH2@FA/PEG/TCPP, toward The cellular uptake of UCNP conjugates into HeLa cancer cells was visualized by Bio-TEM and HeLa cells were evaluated through an MTT assay under normoxic and hypoxic conditions. HeLa CLSM. As shown in Figure 9A, UCNPs@SiO -NH @FA/PEG/TCPP was internalized into the cells in cells were treated with different concentration2 s (0, 21, 5, 10, 20, 30 μg/mL) of TCPP-containing samples 10 min (red fluorescence for conjugated TCPP). This result indicates that the UCNPs are taken up that were previously normalized as free TCPP, and received 660 and 808 nm laser irradiation for free by the cells, presumably due to folic acid-mediated endocytosis and small size-based nonspecific TCPP and UCNPs, respectively. As shown in Figure 6, in the absence of laser irradiation, all uptake [55]. To confirm the internalization of UCNPs@SiO -NH @FA/PEG/TCPP in HeLa cells, cells experimental groups showed a low cytotoxicity regardles2s of t2he TCPP concentration and oxygen were photographed by Bio-TEM (Figure 9B). The diameter of HeLa cells was estimated to be 20–40 μm, presence. UCNPs@SiO2-NH2@FA/PEG (without TCPP) displayed an insignificant cytotoxicity either and UCNPs@SiO -NH @FA/PEG/TCPP was scattered inside or near cells, similar to the original shape with or without2808 n2m light regardless of oxygen due to no lack of singlet oxygen generation. of UCNPs@SiO -NH @FA/PEG/TCPP. In addition, the uptake of UCNPs@SiO -NH @FA/PEG/TCPP However, UCN2Ps@S2iO2-NH2@FA/PEG/TCPP appeared to generate singlet o2xyge2n due to a clear was observed in an in vitro spheroid model system mimicking tumor tissues in vivo (Figure 9C). cytotoxicity. In fact, UCNPs@SiO2-NH2@FA/PEG/TCPP killed ~80% of HeLa cells at 30 μg/mL under The Z-stack images of spheroids were obtained from slices with a 20-μm step size and showed 808 nm laser irradiation. The overall cytotoxicity degree of UCNPs@SiO2-NH2@FA/PEG/TCPP plus red fluorescence. Based on the spheroid result, UCNPs@SiO -NH @FA/PEG/TCPP was predicted the 808 nm laser was almost the same as that of free TCPP plus2the 6260 nm laser, which indicated the to penetrate into tumors in vivo. Despite the absence of in vivo tumor ablation, we believe that effective upconversion of our sample for tumor ablation. Nevertheless, the cytotoxicity of these overall in vitro data demonstrate the great potential for tumor targeting and penetration of our UCNPs@SiO2-NH2@FA/PEG/TCPP (70% cell viability) was significantly reduced under hypoxic UCNPs platform. conditions. Figure 66.. CyCtyototxoicxitcyity eveavlaulautaiotinon ofof frferee TCPPP,, UCNP@ SiO2@FA//PEG,, and UCNP@ 2 SiO2@FA/PEG/TCPP under normoxic or hypoxic conditions with or without laser irradiation (660 nm, 2 22 30 min, 10 mW/cm2 for free TCPP; 808 nm, 30 min, 1.5 W/cm2 for others). Data are presented as means ± SDs (n = 7). * p < 0.003 over Laser(−) and ** p < 0.001 over Laser(−). ± SDs (n = 7). * p < 0.003 over Laser(−) and ** p < 0.001 over Laser(−). 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 thePDF Image | Red LED Erbium Upconverting Nanoparticles v Cervical Cancer
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