3. Results and Discussions
3.1 Synthesis of fluorescent ZnO NPs and optimization of
fluorescence: A facile chemical method was employed to synthesize the
fluorescent zinc oxide NPs as represented in Figure 1 . A LSCM
was employed to perform a detailed study on the optical properties of
the synthesized fluorescent particles. First, we performed XYZʎ scanning
in confocal microscopy, which suggested that these particles can be
imaged using excitation at 405 and 488 nm with an emission wavelength in
the range of 415-460 and 504-668 nm, respectively
(Supplementary Figure S3 ). Since lower wavelength is
not suitable for live imaging for longer period of time, 488 nm was used
as the excitation wavelength for all the nanoparticle retention studies.
Figure 2(a) and (b) show that fluorescence intensity
increases with Tween-80 concentration. Since we aim to retain
fluorescence till 72hour, 20% (v/v) Tween-80, was selected for further
investigations. Additionally, to retain the fluorescence in DMEM media
and to obtain a slower release, the ZnO_T particles were coated by PLL.
The dispersion of the ZnO_T and ZnO_T_PLL NPs in DMEM media along the
course of 72 hours has been shown in Figure 3(a) and(b) . The time course of fluorescence intensities, as shown inFigure 3(c), demonstrated a significantly more retained
fluorescence (p<0.0001) of the ZnO_T_PLL NPs over ZnO_T
NPs. The result clearly shows that PLL encapsulation is able to retain
the particle fluorescence level for a longer period of time. In order to
justify the ability of the NPs to be stored and transported, the
fluorescence of dry ZnO_T_PLL NPs was monitored over a period of one
year, as shown in Supplementary Figure S4 . The particles were
found to retain their fluorescence over three months, showing a
potential for transportation and storage.
3.2 Characterization of NPs: FESEM images
(Figure 4(a) and (b) ) and TEM images (Figure
4(c) and (d) ) show that the resultant NPs had a roughly
spherical shape with size average size of 21 nm and 29 nm for ZnO_T and
ZnO_T_PLL respectively. In order to obtain the size distribution, DLS
analysis was performed. The results reveal the average hydrodynamic
diameter of 73.05 nm and 104.82 nm for uncoated (ZnO_T) and coated
(ZnO_T_PLL) NPs, respectively (Figure 4(e) and (f) ).
EDX spectra (Figure 5(a) and (b) ) indicate the presence of Zn,
O, and C both in ZnO_T and ZnO_T_PLL due to the presence of ZnO and
Tween-80. The introduction of N in ZnO_T_PLL attests to the presence
of PLL in the NP.
The FTIR analysis shown in Figures 5(c) and (d)confirms the peak at around 470 cm-1, which
corresponds to the stretching vibration of the Zn-O bond present in all
the NPs (Khan et al., 2010).
The absorption peak at 1450 cm-1 from the spectra of
ZnO_T is due to bending vibration of O-H, which may be accounted for by
the interaction of ZnO with the C-H bond of Tween-80
( ). A broad peak at 3400
cm-1 in ZnO_T suggests an abundance of O-H groups
present on the surface of ZnO_T. The addition of a peak at 1590
cm-1 in the ZnO_T_PLL spectra is due to the
introduction of amide groups present in PLL. The broad peak from
3100-3600 cm-1 is due to overlapping of amino and
hydroxyl groups, the former introduced from PLL
(Babic et al., 2008). The
surface charge of ZnO_T and ZnO_T_PLL particles were measured by the
zeta potential assay. Supplementary Figure S5 clearly depicts
the variation of the surface zeta potential for a wide range of pH. The
result shows that the surface charge of ZnO_T_PLL (4.6 mV) was found
to be higher than ZnO_T (-0.5 mV) at pH 5.5, indicating the greater
potential of ZnO_T_PLL to be attached to the cell surface.
3.3 NP internalization and retention study: In order to
evaluate the efficacy of drug uptake and retention of ZnO_T_PLL NPs,
they were tested on MCF-7 cells. The MCF-7 cells were chosen to mimic
epithelial uptake in cells from the mammary gland, which is the cell
type that is generally targeted for breast cancer treatment. Since LSCM
coupled with an incubator can be employed for time-lapse 3D imaging,
here we implement LSCM based imaging set-up for in-vitro testing
of particle retention and ROS accumulation in tumor cells. The schematic
diagram for the investigation on the particle internalization, cellular
retention, and ROS generation is shown in Figure 1. In order to
depict the interaction between the particles and cells, we performed
time-lapse imaging for 5 hours using DIC mode after incubation for two
days. This video demonstrates the dynamics of MCF-7 cells and consequent
changes in cell morphology with time when treated with ZnO_T_PLL
(Supplementary Video S1) . However, such a continuous time-lapse
generation for three days was avoided as a long-exposure to laser may
lead to cell apoptosis.
Although Supplementary video S1 was able to capture the dynamic
changes in cells, such imaging cannot be used for quantification of
retention of ZnO_T_PLL. In order to image the particle internalization
along with the cell height, fluorescent intensity was captured for 38
stacks in order to cover the top-most and bottom-most planes, which
varies in a range from 0 to 38µm. Specifically, 3D imaging was performed
to obtain the integration of fluorescent signals from individual planes
across the layers within a cell. The specific advantages of 3D imaging
is shown in Figure 6 through a comparison of 2D and 3D images.
It is evident from Figure 6(c) and Supplementary Video
S2 that the internalized particles were present at multiple planes. The
individual z-scans spanning a depth of 38µm were created by compression
of individual 2D images presented in Supplementary Figure S2(i)and (ii).
Similarly, ROS generation at different z-planes are shown inSupplementary Figure S2(b) . The result shows how the 3D
reconstruction provides accurate signals corresponding to the amount of
internalized particles, and ROS generation, when compared to 2D imaging,
thereby making the quantification more reliable (Figure 6(a) and
(b) ). We also present the depth coding for all the cells with
internalized particles in Figure 6(d) that clearly indicates
that MCF-7 cells are present at different z levels, confirming the
necessity of 3D imaging. To further attest to the internalization of the
particles inside MCF-7 cells we combined fluorescence imaging and DIC
imaging and the merged image in Supplementary Figure S6 shows
that the ZnO_T_PLL particles are present inside the cells and not on
the surface of the cells.
Since NP retention and drug-mediated cell death is known to be crucial
in determining the efficacy of the NP formulation, first, we focus on
the investigation of the cellular retention. MCF-7 cells were incubated
with 40 µg/mL of ZnO_T_PLL NPs, and the imaging of particle
internalization was acquired over 72 hours. Figure 7(a),(b)shows the representative images of particle retention at 0, 12, 24, 36,
48, and 72 hours along with intensity mapping. Since NPs are known to
induce apoptosis through the generation of ROS in excess amounts, we
focus on investigating the dynamics of the ROS produced in assessing
this as the mechanism to induce toxicity in the presence of ZnO_T_PLL
NPs. Figure 7(c) and (d) show the time-lapse
images corresponding to intracellular ROS generation at various time
intervals and corresponding intensity maps in a cell population.Figure 7(e) represents the merged images of cellular
internalization and ROS formation.
Specifically, 40 µg/mL concentration ZnO_T_PLL was chosen for this
study since the particle internalized was found to be significant at 48
hours for this concentration. The comparison of particle internalization
in MCF-7 cells at 0, 12, 24, and 48 hours in the presence of 10 and 40
µg/mL of ZnO_T_PLL NPs and corresponding intensity maps are shown inSupplementary Figure S7 . The confocal images of the cells
showed a dose-dependent fluorescence intensity with a significantly
higher fluorescence signal at 40 µg/mL. The comparative study suggests
the suitability of using 40 µg/mL as the concentration to be assessed.
Moreover, we performed a study on differential cell toxicity and the
preferential killing capability of the ZnO_T_PLL NPs for MCF-7 cells
over healthy L929 cells. Supplementary Figure S8 shows
that the cell viability of MCF-7 cells was significantly
(p<0.0001) lower than normal L929 cells in the presence of
ZnO_T_PLL NPs (p<0.0001). The viability of MCF-7 cells was
found to be 43.53%, while L929 cells were 83.3% viable post 24 hours
of incubation. The results clearly demonstrate the preferential killing
of cancerous MCF-7 cells over healthy L929 cells. This study also showed
that 40 µg/mL could be used as a subtoxic dose as the normal cells show
significantly higher viability (p<0.0001) than cancer cells
over a period of 72 hours (Supplementary Figure S8 ).
Next, we show how LSCM allowed internalization study in single cells
through enabling 3D imaging at a higher resolution, which ensured the
internalization of the particles in subcellular parts rather than
particles being adsorbed on the cell surface. Figure 8 (a) and
(b) show the internalization of ZnO_T_PLL in single cells.The result also shows the aggregation of ZnO_T_PLL NP clusters to be
localized in the outer cell membrane of the MCF-7 cells up to 12 hours
as found in Figure 8(ii) with no significant morphological
changes in the cells. Detectable fluorescence at 12 hours post-treatment
indicates the starting of ZnO_T_PLL internalization inside the
cytoplasm of the MCF-7 cells (Figure 8(a),(b) ). It can be
concluded that, early phases of ZnO_T_PLL internalization inside the
cytoplasm of the cell is not associated with a prominent change in
morphology (Figure 8(iii) and Supplementary Figure S9 ). The
presence of higher levels of fluorescence at 48 hours, as evident from
images of cellular uptake study, affirms the presence of the ZnO
particles in the second day of treatment, which further confirms the
cellular retention Figure 8(v) . The result shows that LSCM,
being a potential tool to evaluate the NP internalization dynamics, can
be employed to get an insight into the delivery of ZnO_T_PLL into
MCF-7 cells for 72 hours.
Next, we present the simultaneous measurement of ROS in single cells at
different times. Figure 8(c),(d) shows the intensity of MitoSOX
dye and corresponding spatial intensity mapping in single cells. The
results show that the ROS formation was rather sporadic up to 12 hours
of treatment, owing to the lower amount of NPs present in the cells
(Figure 8(c) and (d) ). A modest increase in ROS formation was
observed after 24 hours of treatment. In contrast, at 36 hour, the
abundance of red spots indicates a significant increase in ROS
production likely due to accumulation of ZnO_T_PLL NPs in the
cytoplasm (Figure 8(c) and (d) ). The result clearly shows that
The NP internalization and ROS accumulation at 72 hours corresponds to
complete destruction in cell morphology as a result of cell death(Figure 8(vi)). . From the above results, it can be concluded
that the use of laser scanning helped us to gain a better insight not
only in deciphering the internalization dynamics but also to identify
the spatial distribution of particles inside single cells. Such spatial
distribution study in single cells indicates a strong interdependence
between ZnO_T_PLL particle retention and ROS generation in NPs.
3.4 Time course of cell viability for 72 hours: In order to
assess the correlation between NP internalization, ROS formation, and
cell death in a heterogeneous cell population, a live-dead imaging assay
was conducted at 40 µg/mL concentration at the same time points, i.e.,
0, 12, 24, 36 and 72 hours (Figure 9 ). As evident fromFigure 9 , early time points up to 12 h (cell viability of
78.41%) show fewer cells with PI uptake, attributed to the lower uptake
of NPs. The results show that following 36 h, the percentage of cells
with PI uptake increases and gets associated with the disruption of
membrane integrity (viability=16.78%). Hence, it can be concluded that
significant toxicity (cell viability= 5.02%) towards tumor cells can be
induced at 48 h through the application of 40µg/mL ZnO_T_PLL particles
(Figure 9 ). Such ROS-mediated cell death can be attributed due
to the capability of ZnO NPs in inducing ROS like hydroxyl and
superoxide radicals attributed to their semiconductor properties and to
perturb electronic transfer processes in the cell
(Ancona et al., 2018)).
3.5 Quantification of NP internalization, ROS generation, and
cell viability: Since the imaging results indicated the interdependence
between ZnO_T_PLL particle retention and ROS generation, we further
performed quantification of NP retention, ROS generation, and cell
viability dynamics. The time course of particle retention and percentage
increase in ROS has been depicted in Figure 10 (a) and (b) . The
percentage cell viability of MCF-7 cells with respect to time has been
depicted in Figure 10(c) . The quantification of cellular
internalization, ROS accumulation, and cell viability was performed
based on numerous images taken simultaneously at a single time point.
The result suggests a sigmoidal response for internalization of
ZnO_T_PLL NPs with time. The results also indicate that it is possible
to develop an in vitro assay for assessing the efficacy of the
nanoformulation as an anticancer agent through the proposed imaging set
up.
Additionally, we performed the fitting of various functions and found
that a pseudo-first-order kinetic model can be fitted to the
internalization of particles with time (Figure 10(a) ). A
positive Pearson’s correlation value between percentage particle
internalization and ROS generated (r= 0.942) shows that there is a
strong correlation between ROS generation and particle internalization.
On the other hand, negative Pearson’s correlation values between
percentage particle internalization and cell viability (r = -0.9838) and
ROS generation and cell viability (r = -0.98175) indicate the synergy
between long term particle retention and toxicity towards tumor cells.
The time-dependent analysis of intracellular events indicates a possible
mechanism of apoptosis through mitochondrial ROS generation in cells
when invaded by ZnO_T_PLL NPs. A schematic of these intracellular
events has been shown in Figure 10(d) . Overall, it can be
concluded that the confocal microscopy assisted investigation of
mitochondrial ROS production inside the MCF-7 cells can also be used for
deciphering mechanisms underlying the intracellular events.
4. Discussion
Fluorescent nanoformulations have been an attractive research topic in
the field of cancer research both for imaging and therapeutic purposes.
Specifically, ZnO NPs are one of the potential anticancer agents against
MCF-7 cells and can be used for treatment of breast cancer
(Sadhukhan et al.,
2019; Hong et al.,
2011; Ma et al., 2015;
Sureshkumar et al., 2017;
Gupta et al.,
2015; Kavithaa et al.,
2016; Salari et al.,
2020; Boroumand Moghaddam et
al., 2017; Farasat et al.,
2020; Lestari et al.,
2018; Wahab et al., 2014).
However, quantitative imaging of spatial distribution of NPs in living
cells over days was not investigated much. A detailed study on cell-NP
interaction using time-lapse microscopy assumes importance in assessing
the toxicity as well as therapeutic potential. In this paper, we
performed optimization of the NP synthesis to achieve the fluorescence
that can be used for 3D imaging for three days in cell culture medium.
In order to achieve this, Tween-80 assisted fluorescent ZnO NPs were
coated with PLL. The fluorescence in this case might be attributed to
the chemically bonded oxygen molecules of the Tween-80. These act as
scavengers of the photogenerated electrons and transfer them to deep
traps. These electrons upon recombination with trapped holes may result
in a recombination centre for visible emission
(Khan et al., 2010). When
characterized, there was a disparity in the aerodynamic and hydrodynamic
diameter of the particles, which could be accounted to the fact that ZnO
being an amphoteric oxide, undergoes hydrolysis in water. This results
in the formation of a hydroxide coating, ultimately leading to physical
adsorption of water molecules on the surface
(Wang et al., 2017).
As evident from the increased hydrodynamic diameter in ZnO_T_PLL NPs,
it can be concluded that PLL coating increased the hydrophilic nature of
the NPs. Hanley et al. demonstrated the preferential uptake of ZnO NPs
in cancerous cells compared to healthy T cells
(Hanley et al., 2008). It
has also been hypothesized that the hydrophilicity of the synthesized
particles may remain beneficial for passive targeting owing to its
ability to escape macrophage capture
(Allahverdiyev et al.,
2018). Although future experiments need to be conducted, it can be
expected that ZnO_T_PLL NPs being hydrophilic may overcome the threat
of macrophage capture generally found in lipid and polymeric particles.
There is limited information on hydrolysis of zinc-oxide inside
lysosomes due to the acidic pH of cancer cells during long-term imaging
( ; Xia et al., 2008) .
Although further imaging experiments need to be performed to investigate
on this matter using lysosomal staining
(Dong et al., 2018; Xia
et al., 2008), in this work, we performed the measurement of zeta
potential to check the stability of the synthesized zinc oxide material
over a range of pH . Our results showed that the PLL coating leads to an
improved range of zeta potential which provides a roadmap to synthesize
a reasonably stable particle. At the same time the zeta potential
remains in the favorable range for the attachment of the ZnO particles
on the surface of MCF-7 cells. Poly-L-lysine being a cationic polymer,
it exerts a layer of positive surface charge. However, the stability of
the particle can be further improved by coating the fluorescent ZnO with
a thicker PLL layer by altering the PLL concentration while coating the
particles. This may lead to a tailored particle that is stable as well
as suitable for faster attachment to the cell surface.
Our current study focuses on imaging of MCF-7 cells at 40 µg/mL of
ZnO_T_PLL upto 72 hours. However, a future work is rather needed for a
detailed study on retention dynamics at various concentrations of ZnO
NPs. Since it has been found that the fluorescent ZnO nanostructures in
the range 100-400 nm induce preferential killing in MCF-7 cells compared
to L929 cells at 40 µg/mL, we chose to perform the 3D imaging study at
this concentration. Additionally, a co-culture study was performed to
show that normal T cells can be maintained at 85% viability in the
range of 10-40 µg/mL concentration of FITC-tagged fluorescent ZnO
particles, whereas cancerous T cells were found to be less than 10%
viable at 40 µg/mL NP concentration
(Hanley et al., 2008).
Also, there are multiple studies on non-fluorescent ZnO particles for
which the viability of normal cells (HBL100 and MCF10A) were found to be
significantly higher than MCF-7 cells in presence of ZnO nanostructure
(Kavithaa et al.,
2016; Farasat et al.,
2020). It has been shown that 60% viability of HBL100 cells can be
retained within 48 hour using ZnO nanorod of size range from 70 nm to
140 nm at a concentration of 10-20 µg/mL
(Kavithaa et al., 2016).
Based on the concentration range and particle size of the existing ZnO
NP experiments (Table S1), here we choose 40 µg/mL to track the NP
distribution and ROS generation while maintaining lower cytotoxicity to
normal cells. Although, a comparison of cell viability between MCF-7 and
L929 in presence of the synthesized NPs shows a preferential killing of
the MCF-7 breast cancer cell line (Supplementary Figure S8 ),
further optimization can be done for minimization of toxicity towards
healthy cells.
Although generation of ROS is taken as a mechanism for inducing toxicity
in cancer cells, there is limited data on spatiotemporal ROS dynamics
induced by ZnO particles in MCF-7 cells
(Gupta et al., 2015).
Previously, Gupta et al.
(Gupta et al., 2015) has
shown the evidence of ROS generation with varying FITC tagged ZnO NP
concentration at 24 hr, but the correlation between time course of
particle internalization and ROS formation was not evident.
One of the limitations of the current study is that it focuses on
monitoring of cell viability in individual cultures of MCF-7 and L929
cells. However, a comparison cell-NP interaction in a co-cultured MCF-7
and L929 cells will be required for gaining insight on the preferential
internalization of these particles in the MCF-7 cells. Similarly, the
formation of ROS generated as a result of oxidative phosphorylation can
be monitored in normal cells as well as tumor cells inside the
co-culture model. In order to understand the molecular interaction, a
mechanistic model can be obtained for simultaneous prediction of
internalization, ROS and viability.
Previously, it has been shown that fluorescent ZnO acts as a promising
therapeutic agent for reducing the tumor volume from 1.2cc to 0.6cc in
15 days (breast tumor) in rat models
(Hong et al., 2015). Very
recently, non-fluorescent ZnO has been tested previously on rat models
and has been seen to successfully reduce breast cancer tumor volume
(Tanino et al., 2020; .
Since the proposed work forms a solid basis for choosing ZnO particles
as a potential candidate for treating breast cancer through in
vitro imaging assay, we propose that the PLL coated ZnO NPs can further
be tested in vivo in a rat model. Some of the recent
investigations have revealed that ZnO can be effective as
anti-proliferative agent and inhibition agent against various viral
strains such as H1N1 and simplex viruses
(Ghaffari et al.,
2019; Tavakoli et al.,
2018; Abdul et al., 2020;
Faten & Ibrahim, 2018).
Hence, future studies can also be conducted on the effectiveness of
ZnO_T_PLL as an antiviral agent.