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)