Abstract
Generally, investigations on nanomedicine involve conventional imaging
techniques for obtaining static images on nanoparticle internalization
at a single time point where various phases can be overlooked. In
contrast, 3D live-cell imaging can be used for obtaining cellular
retention of drugs at various phases, and cells can be followed for
days. This article demonstrates the application of time-lapse
microscopy in the investigation of Poly-L-lysine coated ZnO nanoparticle
dynamics. In this work, a laser scanning confocal microscope has been
employed to quantify the dynamics of internalization particles and
reactive oxygen species generation (ROS) using volumetric
imaging. Firstly, we show that simultaneous spatial mapping of
nanoparticle uptake in MCF-7 cells and ROS in a single cell can be used
to identify the interdependence between the accumulation of particles
and ROS generation. Secondly, monitoring of ROS formation and
cytotoxicity using the same imaging platform offers an advantage over
monitoring these parameters using various instruments. Finally, the
ability of the fluorescent particles in inducing a significant reduction
in cell viability suggests its potential to be used as a therapeutic
agent. The proposed framework opens up a new avenue of research for
investigating mechanistic aspects of ZnO particle adsorption in
vitro through long term imaging.
Keywords : Fluorescent ZnO particle, Time-lapse microscopy, 3D
Live-cell imaging, laser scanning confocal microscope, Reactive oxygen
species
Introduction
Breast cancer treatment includes various modalities like surgery,
radiation therapy as well as drug therapy for the prevention of
metastasis. Since these drugs have prominent cytotoxic effects on both
cancerous and healthy cells, there is a significant effort in developing
biocompatible nanoformulations that can be tailored to their intended
applications as therapeutics
(). Determination of
bioavailability and toxicity of such nanoparticles (NPs) require the use
of imaging approaches, ideally with 3D capabilities. Monitoring cell-NP
interaction for tumor cells using high-resolution imaging may provide
insights on the attachment of particles on the cell surface
(Song et al., 2015;
Wang, Lee, Kim, & Zhu,
2017; Xia et al., 2008;
Kiefer et al., 2020). In
order to quantify the amount of internalized particles, it is crucial to
perform imaging of various z-sections and perform 3D reconstruction of
fluorescent intensity using confocal microscopy. Recent advancement in
3D visualization of nanoparticles distribution in living cells and
tissue involves volumetric imaging using laser scanning confocal
microscopy (LSCM) (Dias,
Werner, Ward, Fleury, & Baulin, 2019; Ramos-Gomes, Ferreira, Kraupner,
Alves, & Andrea Markus, 2020).
Drugs encapsulated in liposomes and polymeric NPs were found to be under
clinical trials for the treatment of breast cancer
( ). In contrast,
metal-based nanosystems, including gold, magnetic NPs, are still in the
preclinical stage therapeutics
( ). Although there are
some investigations focusing on the preliminary evaluation of ZnO
toxicity in MCF-7 cell line (Supplementary Table S1 )
(Sadhukhan et al., 2019;
;
;
Sureshkumar, Jothimani,
Sridhar, Santhosh, & Venkatachalapathy,
2017; ), there are limited
data generation focusing on retention dynamics of ZnO particles. One of
the major challenges in generating preclinical data is to develop
particles that could be used for 3D imaging in live cells for a
prolonged period of time
(Dias et al., 2019;
Ramos-Gomes et al., 2020). Here we demonstrate the synthesis of a
fluorescent ZnO NP with Poly-L-lysine (PLL) coating that can be imaged
for a longer period using confocal microscopy and also facilitates
preferential uptake in tumor cells. These NPs were specifically tailored
to exhibit the fluorescence at 488 nm through the addition of an optimal
amount of Tween-80, and imaging of particle internalization was
performed up to 72 hours. To quantify the amount of internalized
particles in cells, we perform imaging of various z-sections and 3D
reconstruction of fluorescent intensity.
Zinc oxide NPs have been shown to be effective in killing cancer cells
for a wide range of cancer cell lines and reducing tumor size
(Hussein & Ministry of
health –clinical pathology center, 2017; Tanino et al., 2020;
Sivakumar, Lee, Kim, & Shim,
2018). Additionally, it has been used for various biomedical
applications due to its long proven biocompatibility
(; . Some form of
Zn-based particles has been used as antiviral compounds that are known
to be effective against the virus
(Ghaffari et al.,
2019; Tavakoli et al., 2018;
Abdul, Muhammad, Ullah,
Asmat, & Abdul, 2020; Faten
& Ibrahim, 2018). Although, multiple investigations show that intact
and modified versions of ZnO can be used to suppress MCF-7 cell growth
and proliferation in vitro(;
; Boroumand Moghaddam et al.,
2017; ;
;
Wahab et al., 2014),
in-depth preclinical studies are required focusing on quantification.
Generally, fluorescent microscopy has been used for studying the
internalization of ZnO particles
( Hong et al., 2011;
Sadhukhan et al., 2019; Ma
et al., 2015). However, 3D
imaging and quantification of particle internalization dynamics for a
longer period are not possible if the particle is not optimized for
imaging with confocal microscopy. There are few attempts to formulate
fluorescent ZnO so that the particle can be monitored through 2D imaging
using confocal microscopy
( Sureshkumar et al.,
2017; Gupta et al., 2015;
Wang et al., 2017;
Xia et al., 2008) . A
summary of fluorescent ZnO particles that can be used for suppressing
breast cancer cell lines are presented in Supplementary Table
S1 .
Recent advancement in the synthesis of multifunctional particles focuses
on intrinsic and extrinsic fluorescent zinc oxide particles for
targeting MCF-7 cells. One of the studies focuses on the synthesis of
fluorescent ZnO nanowires using vapor deposition in a similar size
range, and the internalization in MCF-7 was detected within an hour at a
concentration of 30 µg/mL
(Hong et al., 2011; Ma
et al., 2015). Ma et al. (2015) synthesized fluorescent ZnO quantum
dots that shows that the particles are effective in regulating cell
proliferation. Sureshkumar et al. (2017) synthesized polyquaternium
capped zinc oxide nanodisc and used as an anticancer agent against MCF-7
cells. The synthesized NPs were found to be more stable than the
commercial dyes, but the internalization dynamics was shown only for a
short duration of 6 hours. One of the recent studies on particle
internalization was performed using fluorescence microscopy using 2D
imaging during the early phase of 1.5 hours
(Sadhukhan et al., 2019).
While most of these studies focus on the synthesis of the particle and
viability studies, they do not elucidate the retention dynamics of ZnO
particles and reactive oxygen species (ROS) generation. One of the
studies shows that zinc oxide particles tagged with FITC were able to
induce ROS generation in MCF-7 cells. Cell viability study was performed
in the range of 8-500 µg/mL, whereas the cellular uptake of ZnO was
studied at 100 µg/mL (Gupta
et al., 2015) (Supplementary Table S1 ). Although this study
reveals that ZnO can be used as a potential candidate for inhibiting
MCF-7 cell proliferation and ROS generation, FITC is more prone to
photobleaching, which may hamper the long-term imaging study.
Although ZnO particles can be imaged using 405 nm, excitation at higher
wavelength is crucial to avoid cell apoptosis due to prolonged exposure
to nearly UV light range during 3-day imaging. In order to address this,
we demonstrate the synthesis of ZnO particles, where the concentration
of Tween-80 was optimized to maintain the required level of fluorescence
to be obtained through excitation at 488 nm. The rationale behind
coating of the particles with Poly-L-lysine (PLL) is to enhance the
biocompatibility and resisting photobleaching enabling long-term
monitoring of the fluorescence during live imaging. It has been shown
previously that PLL can be used for increasing the biocompatibility
(Babic et al., 2008;
Marsich et al., 2012),
The existing cytotoxicity studies were performed in the range of 24-48
hours. However, most of the ZnO internalization studies in MCF-7 cells
were performed for approximately six hours using 2D imaging that
provides information on the surface attachment of the particles. For
example, the cellular uptake of FITC tagged ZnO with a size range from
100 - 400 nm was studied for up to 3 hours
(Gupta et al., 2015). Since
the time required for particle uptake can be shortened using smaller
size particles, fluorescent ZnO nanorods in the range of 10 nm were
shown to be internalized within 90 minutes in MCF-7 cells
(Ma et al., 2015).Similarly, internalization of ZnO tagged with fluorescent drugs in the
range of 30-40 nm was studied in MCF-7 cells between 3 and 6 hours
(Sureshkumar et al., 2017;
Sadhukhan et al., 2019). To
the best of our knowledge, there is no investigation focusing on
optimization of a nanostructure that can be used for 3D live-cell
imaging of MCF-7 cells for a prolonged period. In this article, we
demonstrate z-stack imaging of ZnO uptake, ROS formation, and cell fate
imaging for up to 72 hours using fluorescent ZnO.
In order to show that the PLL coated ZnO particle can be used for
simultaneous monitoring of internalization, we present the imaging assay
using laser scanning confocal microscopy (LSCM) attached with a
CO2 incubator. One of the major novelties of the
proposed assay includes the generation of data based on 3D imaging of
ZnO internalization and ROS formation. In order to depict the
spatiotemporal distribution of the particle and the correlation between
NP internalization and ROS production, we have shown the spatial
intensity mapping after merging the z-stacks. While most of the existing
studies on ZnO NP internalization has been performed at 1 - 6 hour range
(Sadhukhan et al., 2019;
Hong et al., 2011; Ma et
al., 2015; Sureshkumar et
al., 2017; Gupta et al.,
2015), the current work focuses on assessing the particle distribution
using 3D imaging for three days. This study demonstrates that PLL coated
particles in the range of 30 nm remain fluorescent over a longer period
of time than fluorescent. Moreover, we show that the particle
internalization, ROS formation, and viability can be monitored in the
same imaging platform to assess the therapeutic potential in MCF-7 cell
lines. Our results demonstrate that 3D imaging using LSCM represents a
promising and powerful approach for preclinical investigations, which
offers advantages of performing high-resolution imaging in living cells
over other methods of volumetric imaging in fixed cells
(Chen et al., 2013;
Gimenez et al., 2016)