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.