2.2 Synthesis of NPs
2.2.1 Synthesis of ZnO_T NPs: The NPs were prepared using 0.25 M zinc nitrate hexahydrate solution, to which Tween 80 was added. The mixture was stirred at 50°C and homogenized, followed by dropwise addition of 0.25 M ammonium carbonate solution. The pH of the reaction mixture was maintained at 10, and the solution was further mixed at 80°C. Particles were washed with ethanol and dried in a hot air oven at 50°C overnight (Khan et al., 2010).
2.2.2 Synthesis of ZnO_T_PLL NPs : The ZnO_T NPs were incubated in 0.1% (w/v) of PLL and kept overnight at 37℃ for 8 hours. The particles were centrifuged and dried at 37℃.
2.3 Controlling fluorescence of NPs using Tween-80: In order to have higher fluorescent signal of the particle, the concentration of Tween-80 in the precursor solution was varied as 8%, 12%, 14%, and 20% (v/v). The dry particles having fluorescence were characterized using LSCM (Leica SP8 microscope, Germany). The XYZʎ scanning was performed to obtain the excitation and emission wavelength range for the ZnO_T_PLL particle with the 40X oil emulsion objective. The coated and uncoated particles were further immersed and ultrasonicated at 20kHz in DMEM for quantification of the fluorescence of the particles with time.
2.4 Characterization of the NPs: The surface morphology and microstructural evaluation of the synthesized ZnO_T and ZnO_T_PLL NPs were investigated by by Field emission scanning electron microscopy (FESEM) ((JEOL JSM-7610F FESEM with JEOL JEC-3000FC coater) and Transmission electron microscope (TEM) (JEM-2100 HRTEM, JEOL, JAPAN with an accelerating voltage of 20keV). The elemental analysis was done with Energy Dispersive X-Ray Spectroscopy (EDX). Dynamic light scattering (DLS) (Malvern Zeta-sizer ZS90 Nano-series (UK)) was used to analyse the hydrodynamic diameter of the particles whereas Zeta Potential (Malvern Zeta-sizer ZS90 Nano-series (UK)) was used to find the surface charge. Fourier Transform Infrared Spectroscopy (FTIR) (PERKIN ELMER SPECTRUM FTIR) techniques helped in confirming the functional groups present and the successful coating of PLL.
2.5 Cell culture: MCF-7/L929 cells were cultured in DMEM supplemented with 10% dialyzed fetal bovine serum and antibiotics. The cells were seeded (cell density of 2.047x104 cells/mL) in 29 mm glass-bottom dishes and maintained with 5% CO2in a humidified incubator at 37 oC.
2.6 Cellular Internalization Study: ZnO NPs at a concentration of 40µg/mL were added to the cells and incubated. Images were captured using LSCM at 0h, 12h, 24h, 36h, 48h, and 72 h after adding NPs using 40X oil emulsion objective with 488 nm excitation. In order to quantify the extent of internalization of NP, the 3D images were merged in one plane, and the summation of fluorescence intensity was quantified using Leica LAS X software (Venkateswarlu et al., 2020). In order to have unbiased sampling, the LAX software was used for random sampling of three regions for various time points (Supplementary Figure S1 ). Various kinetic models were fit into the internalization data, and the pseudo 1st order kinetic model was found to be the best model with highest R2 value of 0.988. Pseudo 1st order kinetic model used is:
Log(If– It) = log(If)-kt ……………………………………………………………………….(1)
Where If is the fluorescence intensity at 72 hours, It is the fluorescence intensity at time t, and If is considered to be the maximum intensity and hence maximum possible internalization.
2.7 ROS Generation Study: MitoSOX, a mitochondrial superoxide indicator, was used for measuring reactive oxygen species which exhibits red fluorescence upon binding with generated superoxide. MCF-7 cells were incubated with 40µg/mL of ZnO_T_PLL NPs for varying time periods. 1ml of 5µM MitoSOX red was added to cells and incubated for 10 minutes. The cells were thoroughly washed with a warm buffer at 37℃, and imaging was performed at an excitation and emission wavelength of 510 nm, and 574-751 nm respectively.
2.8 Cell Viability Study: The percentage of dead cells induced by ZnO_T_PLL NPs was measured using propidium iodide (PI) (excitation and emission at 493 and 636 nm respectively), a red fluorescent nuclear stain that enters only cells with disrupted plasma membranes. The unaffected status of the live cells was verified by detecting the green fluorescent signal due to calcein formation in them. Calcein-AM, a nonfluorescent dye known to permeate to the cytoplasm easily, is converted to a green fluorescent calcein (excitation and emission at 495 and 515 nm respectively) after acetoxymethyl ester hydrolysis by intracellular esterases in live cells. The cells were incubated with 40µg/mL NPs for varying time periods. calcein-AM, and PI were added in a 1:4 ratio to the cells and incubated for 20 minutes. The cells were then washed with a warm buffer at 37℃ and was used for further imaging. Live cells (Green) and dead cells (red) were counted for calculating the percentage of viability using the formula given below.
%Viability = (N/NTotal)*100…………………………………………………………………………………………………….(2) Where N represents the number of live cells, and NTotalrepresents the total number of cells.
2.9 3D reconstruction of MCF-7 cells: Multiple z-stacks corresponding to different focal planes were scanned in order to construct 3D images of cells internalized with fluorescent zinc oxide NPs. The fluorescence intensity of cells was measured by taking a total of 38 z stacks, which provided us the sample thickness varying between 30-40 µm. We performed the quantification of percentage internalization of NPs at different time points using the merged images obtained from 2D images of individual planes (Supplementary Figure S2 ). A summary of the imaging parameters used for imaging using LSCM is shown in Supplementary Table S2 .
2.10 Statistical Analysis: Data were presented as mean ± standard deviation (SD). ANOVA and paired t-test was performed in statistical evaluation by MS-Excel. A p- value below 0.0001 was considered to be statistically significant.