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Figure Legends
Figure 1: The schematic for the workflow for the synthesis of ZnO_T_PLL nanoparticles and measurement of retention dynamics and ROS accumulation using 3D imaging by LSCM.
Figure 2: Fluorescence level in ZnO_T nanoparticles as a function of percentage of Tween-80 (v/v). (a) 3D imaging of particles corresponding to 8%, 12%, 14%, and 20% Tween-80. (b) Bar plot representation of fluorescent intensities for particles when 8%, 10%, 12%, 14%, and 20% Tween-80 was used. Scale bar in all panels represents 200 µm. p<0.0001.
Figure 3: Comparison of ZnO_T and ZnO_T_PLL fluorescence level in DMEM medium for three days. 3D reconstruction of fluorescent images for (a) ZnO_T and (b) ZnO_T_PLL at 0, 12, 24, 48 and 72 hours. (c) The box plot representation of the fluorescence intensities of ZnO_T and ZnO_T_PLL at various time points, including 0, 12, 24, 48, and 72 h. A statistical test was performed (* indicates p<0.0001). Scale bar in all panels represents 200 µm.
Figure 4: Morphology of ZnO_T and ZnO_T_PLL nanoparticles. SEM images of (a) ZnO_T_PLL and (b) ZnO_T, TEM images of (c) ZnO_T_PLL and (d) ZnO_T confirm the nanoarchitecture of the prepared samples. Hydrodynamic size distribution analysis of (e) ZnO_T_PLL and (f) ZnO_T has been depicted by DLS. Scale bar in all panels represents 50 nm.
Figure 5: Elemental analysis of (a) ZnO_T and (b) ZnO_T_PLL nanoparticles by using EDX. FTIR analysis of (c) ZnO_T and (d) ZnO_T_PLL nanoparticles show the various functional groups present.
Figure 6: Comparison between individual z-stack (2D) and compressed 2D images from multiple stacks (3D). (a) Compressed 2D images for MCF-7 cells obtained from multiple stacks, (A) DIC images, (B) internalized ZnO_T_PLL, and (C) reactive oxygen species after 36 hours of incubation with nanoparticles. (b) Individual z-stack images for MCF-7 cells, (A) DIC image, (B) internalized ZnO_T_PLL, and (C) reactive oxygen species after 36 hours of incubation with nanoparticles. (c) 3D reconstruction of (A) internalized ZnO_T_PLL, (B) ROS in MCF-7 cells. (d) ZnO_T_PLL internalization and subsequent depth coding to demonstrate the presence of cells at different z levels. Scale bar in all panels represents 100 µm.
Figure 7: Simultaneous mapping of cellular retention of ZnO_T_PLL and ROS generation in MCF-7 cell population using LSCM. Incubation of cells with ZnO_T_PLL was performed in the incubator attached with the microscope. Time-lapse images for ZnO_T_PLL Internalization and ROS generation are presented in the form of compressed z-stacks. (a) Cellular retention of ZnO_T_PLL nanoparticles during incubation MCF-7 cells with 40µg/mL of nanoparticles, (b) Spatial fluorescent intensity mapping of the internalized particles at 0,12, 24, 36, 48, and 72 hours. (c) Time-lapse images of mitochondrial ROS (stained with MitoSOX Red) at 0,12, 24, 36, 48, and 72 hours. (d) Spatial fluorescent intensity mapping of ROS generation. (e) Merged window representing both internalized Zno_T_PLL and ROS generation. Scale bar in all panels represents 100 µm.
Figure 8: ZnO_T_PLL induced ROS production in single cells during incubation with 40µg/mL of nanoparticles. Time course of (a) ZnO_T_PLL internalization, (b) Spatial intensity map of internalization, (c) mitochondrial ROS generation and (d) Spatial intensity map of ROS generation at 0, 12, 24, 36, 48 and 72hours. Imaging was performed by laser scanning confocal microscopy after staining with MitoSOX Red. Spatial intensity map was generated from compressed 3D images of ROS accumulation by Image-J software to demonstrate the varying concentration of mitochondrial ROS production. The relative concentrations are depicted by the color intensity with a legend indicated at the right side of the figure. Scale bar in all panels represents 20 µm.
Figure 9: Effect of ZnO_T_PLL nanoparticles on MCF-7 cell viability. (a)-(e) Time-lapse images MCF-7 cells incubated with 40µg/mL of nanoparticles at 0, 12, 24, 36, 48 and 72 hours. Live-dead cell assay was performed by staining with calcein (green) and propidium iodide (red), respectively, where green and red denote live cells and nucleus of dead cells obtained. Scale bar in all panels represents 750 µm.
Figure 10: Correlation between the temporal dynamics of nanoparticle internalization, ROS accumulation, and cell viability. Time course of (a) ZnO_T_PLL internalization in MCF-7 cell, (b) Percentage ROS generation, (c) cell viability of MCF-7 cell, (d) Schematic of a hypothetical pathway for ROS generation facilitated by ZnO_T_PLL and consequent cell death in MCF-7 population.