Elucidating the Uptake and Distribution of Nanoparticles in Solid Tumors via a Multilayered Cell Culture Model
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Multicellular layers (MCLs) have previously been used to determine the pharmacokinetics of a variety of different cancer drugs including paclitaxel, doxorubicin, methotrexate, and 5-fluorouracil across a number of cell lines. It is not known how nanoparticles (NPs) navigate through the tumor microenvironment once they leave the tumor blood vessel. In this study, we used the MCL model to study the uptake and penetration dynamics of NPs. Gold nanoparticles (GNPs) were used as a model system to map the NP distribution within tissue-like structures. Our results show that NP uptake and transport are dependent on the tumor cell type. MDA-MB-231 tissue showed deeper penetration of GNPs as compared to MCF-7 one. Intracellular and extracellular distributions of NPs were mapped using CytoViva imaging. The ability of MCLs to mimic tumor tissue characteristics makes them a useful tool in assessing the efficacy of particle distribution in solid tumors.
KeywordsGold nanoparticles Tumor Multicellular cell layers Tissue
The MCLs developed by Wilson and his colleagues provide a quantitative method that permits direct assessment of drug penetration through solid tissue [17, 18]. MCLs share several properties with solid tumors derived from the same cell type, including a similar but not identical extracellular matrix (ECM) and tight junctions between epithelial cells . In addition, MCLs have been shown to exhibit areas of hypoxia, necrosis, as well as nutrient and proliferation gradients, and ECM generation [20, 21, 22]. The development of MCL models has facilitated quantification of drug penetration through solid tissue. Although the direct in vivo assessment, when feasible, has the advantage of duplicating the clinical environment most closely, in vitro techniques offer the advantage of being able to examine the distribution of agents of interest in the absence of complicating factors such as pharmacokinetics and hepatic metabolism which often differ between mice and humans .
2 Materials and Methods
2.1 Synthesis of GNPs
The GNPs were synthesized via the reduction of HAuCl4 by sodium citrate, which is more commonly referred to as the Turkevitch method . By varying the amount of sodium citrate, the method can yield NPs of varying sizes. In this study, 20-nm particles were chosen since our future goal is to use these NPs for gene delivery. The GNPs were characterized by transmission electron microscopy (H7000; Hitachi Corp. Tokyo, Japan), UV-spectroscopy (Lambda 40; PerkinElmer, Waltham, MA), and by dynamic light scattering using 90 Plus Particle Sizer Analyzer (Brookhaven Instruments Corp. New York, NY) to determine the size of the particles.
2.2 Growth of MCLs
The growth of the MCLs began with the growth of monolayer cells in a 5 % CO2 environment at 37 °C. After reaching confluence, these cells were trypsinized, centrifuged, suspended in media, and counted. Approximately, 150,000–200,000 cells were seeded onto a microporous membrane insert (Millicell, Bedford, MA). After allowing the cells to attach for 2–4 h, the inserts were washed with phosphate buffered saline (PBS) and then suspended in stirred media to grow. With pore sizes of 3 µm, the inserts allowed for the passage of stirred media through the base of the insert as seen in Fig. 2a.
Two breast cancer cell lines were used in this study: MCF-7 and MDA-MB-231. Cells were grown on the MCL insert in Dulbecco′s Modified Eagle′s Medium (LifeTechnologies Inc. Burlington, ON) with 10 % Fetal Bovine Serum (Sigma-Aldrich, Oakville, ON). Figure 2b is an image of an unstained tissue cross-section of MCF-7 cells. The ECM within the tissue was stained with eosin for visualization (Fig. 2c). The thickness of the tissue was controlled by the growth period. MCL incubation with NPs was done by hanging the MCLs in multiwall plates followed by filling the top of the inserts with the GNP and media mixture. A supply of fresh media was placed below the MCL to allow for GNPs that had penetrated the entire MCL structure to diffuse.
2.3 Quantification of GNP Uptake
During incubation, the MCL structures were hung in multi-well plates with a 15 nM GNP/media mixture on the top and a supply of fresh media on the bottom. Both the ‘top’ and ‘bottom’ volumes were collected and measured for gold content via inductively coupled plasma-atomic emission spectroscopy (ICP-AES) (Optima 7300 DV; PerkinElmer, Waltham, MA). By using the known total concentration of GNPs, uptake and penetration through tissue may be measured, which results from taking the difference between the ‘top’ and ‘bottom’ samples. Monolayer cultures were also grown and harvested at three different time points or cell densities. These cultures were incubated with GNPs for 24 h and were used for cell counting and monolayer gold uptake measurement via ICP-AES. To determine the uptake as a function of layers, the difference in uptake between consecutive days of growth was observed (Fig. 8b). Because each new day of growth introduced new layers, the difference in uptake between consecutive days allowed for the quantification of uptake as a function of layer.
2.4 Qualitative Analysis
To qualitatively measure the distribution of the GNPs as well as to provide a measure of MCL growth characteristics, MCL inserts were frozen in OCT compound for sectioning. The frozen MCLs were then sectioned (Cryostat CM1900; Leica, Wetzlar, Germany) into 15–20-µm-thick sections and placed onto slides for imaging. Tissue sections were stained with eosin to show the presence of ECM (Autostainer XL; Leica, Wetzlar, Germany). Stained tissue sections were imaged using the CytoViva Hyperspectral Imaging (HSI) dark-field microscope. By examining the images acquired by HSI (Fig. 8c, d), a qualitative examination of the layer-by-layer penetration was deduced.
2.5 CytoViva Imaging of NP Distribution in MCLs
3 Results and Discussion
In this study, we demonstrate for the first time the differences in GNP uptake between monolayer and tissue-like MCL models. GNPs were used as a radiation dose enhancer, anticancer drug carrier, and an imaging contrast agent in cancer research as discussed in the introduction. However, the success of NP-based imaging and therapy depends on the efficiency of delivery of NPs tumor tissue as illustrated in Fig. 1a. In this study, we investigated the NP transport across tumor tissues by using MCL cell model for the first time. It was successfully used to understand NP diffusion in tissues. The results were consistent with drug diffusion patterns observed in solid tumor in animal models.
It is known that NPs can leak out through the disorganized endothelial cells in tumor blood vessels and enter the tumor tissue (Fig. 1b). In particular, this MCL model was used to study the transport of NPs across the tumor tissue once they leave blood vessels (Fig. 1c). The device used for growing MCLs is shown in Fig. 2a. A tissue cross-section of an approximately 150-µm-thick MCF-7 cell is shown in Fig. 2b and 2c. An unstained tissue cross-section is shown in Fig. 2b, while a tissue cross-section stained with eosin to highlight the ECM is shown in Fig. 2c. We used the CytoViva HSI technique to image the tissue and NPs. Unlike other optical imaging techniques, HSI allows us to map the GNPs via reflectance spectroscopy. This imaging technology does not require optically labeling NPs for their visualization. This is the first time that such imaging technology was used to visualize GNP distribution in tissue-like structures. Figure 3a shows the unmapped dark-field HSI image with GNPs visible as bright yellow spots, while Fig. 3b shows the result of spectral angle mapping on the HSI image. GNPs have been labeled red as a result of matching spectra (shown as an inset figure in Fig. 3c) from individual pixels. Figure 3c shows a few spectra from GNP clusters displayed in Fig. 3a. This imaging technique was used to map NP distribution through the tissue. Our first goal was to investigate the difference between monolayer and MCL cell models in terms of growth before investigating the NP uptake and transport.
There is a tremendous effort to incorporate NPs into existing cancer therapeutic protocols. We have demonstrated that MCL model can be used to study the transport of novel NP-based systems to optimize their delivery to tumor tissue. Our future goal is to study how the size and shape of NPs affect their transport through the ECM using this MCL model. Our preliminary studies showed that smaller NPs display higher tissue penetration as compared to larger NPs (supplementary section S2). This result is consistent with previous in vivo data. For example, Puvanakrishnan et al. investigated the in vivo tumor targeting efficiency of pegylated gold nanoshells (GNSs) and gold nanorods (GNRs) for single and multiple dosing . The results showed that the smaller GNRs accumulated in higher concentrations in the tumor in comparison to larger GNSs. Moreover, Zang et al. have shown that 20-nm GNPs showed significantly higher tumor uptake than 40- and 80-nm GNPs . These in vivo studies clearly show that smaller NPs transport easily through the ECM in comparison to larger NPs. The importance of this study is that we showed that MCL model could be used to mimic NP transport in tumor-like environments. Our results are consistent with published in vivo data. This model can be used to understand the transport and therapeutic response of NP complexes prior to use in animal models. Our future goal is to study how NP size and shape affect their transport in vivo and correlate their transport within the tumor tissue using our in vitro MCL model.
This work demonstrates the importance of understanding the limitations of monolayer cultures in their ability to predict the uptake and effectiveness of cancer treatments in solid tumors. Furthermore, these results underscore the importance of the ECM in terms of throughout tumor tissue. By engaging these issues, GNP-based systems can be designed for combined chemotherapy and radiation therapy to better overcome the resistance to drugs and radiation found in solid tumors . This would accelerate such NP-based innovations into clinics for the improved quality of life of cancer patients.
The authors would like to acknowledge Natural Sciences and Engineering Research Council.
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