Spectra i3x

Confluent HUVEC grown on a 96-well plate coated with 8 μg/ mL collagen and MCF7 cells grown directly on a well plate were exposed to uncoated and PEG-coated ZnO-Dye 847 NPs at different concentrations (10, 20, 50, and 100 μg/mL) for 24 h, each concentration having 4 replicates. Samples were exposed to the NIR source for 2 min, after which 100 μL of 20 mM DCFDA diluted in PBS was added to each well and incubated for 2 h. The fluorescence (E_x/E_m = 495/529 nm) was measured in a microplate reader (Spectra i3x, Molecular Devices) every 15 min for 3 h. H₂O₂ (2%) diluted in EGM-2 and cells in EGM-2 served as the positive and negative controls, respectively, for HUVEC; 2% H₂O₂ diluted in Minimum Essential Medium supplemented with 0.01 mg/mL human recombinant insulin and 10% fetal bovine serum (MEM*, Fisher Scientific) and cells in MEM* served as controls for MCF7 cells.

The irradiation time required to produce the highest level of released ROS was investigated using DCFDA. Four replicates of uncoated ZnO-Dye 847 NPs (10, 20, 50, and 100 μg/mL) were irradiated with the NIR source for 2, 15, and 30 min. As used earlier, 2% H₂O₂ diluted in DI water served as the positive control and DI water served as the negative control. DCFDA diluted in PBS (100 μL of 20 mM) was added to each well and incubated for 2 h. The fluorescence (E_x/E_m = 495/529 nm) was measured in a microplate reader (Spectra i3x, Molecular Devices) every 1 h for 8 h.

The radical scavenging activity of the V. sinaiticum leaf extract was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH) [32 (link)], with a slight modification. Three milliliters of DPPH solution (0.004%) were added to the extract, standard, or blank solution (1 mL). The mixture was incubated in darkness at room temperature for 30 min. The absorbance was measured against the blank using a spectrophotometer at 517 nm (Molecular Devices LLC., Spectra i3x Gangnam-gu, Seoul, Republic of Korea) [33 (link)]. Then, the data were expressed as IC₅₀.