Size-Dependent Gold Nanoparticle Interaction at Nano–Micro Interface Using Both Monolayer and Multilayer (Tissue-Like) Cell Models
Gold nanoparticles (GNPs) are emerging as a novel tool to improve existing cancer therapeutics. GNPs are being used as radiation dose enhancers in radiation therapy as well as anticancer drugs carriers in chemotherapy. However, the success of GNP-based therapeutics depends on their ability to penetrate tumor tissue. GNPs of 20 and 50 nm diameters were used to elucidate the effects of size on the GNP interaction with tumor cells at monolayer and multilayer level. At monolayer cell level, smaller NPs had a lower uptake compared to larger NPs at monolayer cell level. However, the order was reversed at tissue-like multilayer level. The smaller NPs penetrated better compared to larger NPs in tissue-like materials. Based on our study using tissue-like materials, we can predict that the smaller NPs are better for future therapeutics due to their greater penetration in tumor tissue once leaving the leaky blood vessels. In this study, tissue-like multilayer cellular structures (MLCs) were grown to model the post-vascular tumor environment. The MLCs exhibited a much more extensive extracellular matrix than monolayer cell cultures. The MLC model can be used to optimize the nano–micro interface at tissue level before moving into animal models. This would accelerate the use of NPs in future cancer therapeutics.
KeywordsGold nanoparticles Multilayer cellular structures Tissue penetration Hyper spectral imaging Size dependence
In post-vascular tumor tissue, the local environment is much more complex than in monolayer cell cultures due to the presence of the extracellular matrix (ECM). The ECM is a structural and biological support network that arises from proteins and molecules secreted by cells as they grow. The composition and architecture of the ECM vary from one cell line to another since different cells secrete different proteins with different functions. The ECM carries out a number of important functions, enables cell-to-cell communication and cell adhesion, and provides a rigid support structure that gives tissues its shape. This support structure also determines the permeability of tissue to molecules and other particles and sometimes acts as a barrier for transport, as discussed in the next section [7, 8, 9, 10]. Eosin is a florescent dye used for staining proteins in the cytoplasm of the cells and collagen in the ECM. As illustrated in Fig. 1, ECM (marked in green) is not heavily present in between cells compared to tissues. Hence, ECM does not act as a barrier for NP transport in monolayer cell cultures whereas it does for NP transport in tissue-like MLC structures. For example, it was shown that NP penetration into the core tumor spheroids diminished as particle sizes increased from 20 to 100 nm NPs . Several factors have been proposed to explain the poor penetration and distribution of GNPs in solid tumors. These factors include the binding of GNPs to tumor tissue, increased interstitial fluid pressure, and hindered diffusion due to cell packing and the ECM [7, 8, 11, 12, 13]. Furthermore, the elimination of the pressure gradient in solid tumors yielded only minimal gains in macromolecule delivery which suggests that cell packing and ECM are major factors in the differences between monolayer and solid tumor penetration dynamics [8, 12, 14].
The geometry of the MLCs also sets it apart from other 3D cell cultures, such as the multicellular spheroid, as discussed in the previous section. In the spheroid geometry, drugs and agents pass radially inwards from the edges of the spheroid. This geometry is often the reverse of in vivo physiology and makes it difficult to ascertain the penetration of drugs in a solid tumor. The MLC provides a top-down or side-to-side geometry and better mimics the distribution of agents out of a blood vessel and down into tissue [15, 16, 17]. In addition, it has been reported that within tumors, cells can appear further than 150 µm from the nearest blood vessel . In such a case, any agents delivered to the tumor through the bloodstream will have to navigate in the heterogeneous tumor environment without assistance from any vascular network to reach their target cells. Our proposed MLC model can also be used to examine the penetration and transport of GNPs through such tissue.
While in vivo tumors offer the most clinically relevant tool for determining the spatial distribution and penetration of GNPs, the complex biological factors prevent a focused analysis of the individual components that contribute to the transport of GNPs and other agents [19, 20, 21]. Using this MLC model, the limiting effects of the ECM, the thickness of cell layers, and the GNP transport mechanisms of GNPs in tumor tissue can be measured in a less complex biological microenvironment. In this study, we investigated the size dependency of NP transport in tumor tissues using the MLC model for the first time. We were able to image the penetration of NPs in these tissue sections for the first time. The accumulation of GNPs in the MLC tissue was measured to determine the major factors affecting GNP transport through tumor tissue in the absence of vasculature. Hence, the MLC model can be used to improve the design of NP-based systems for improved outcomes in imaging and therapeutic applications in the field of nanomedicine.
2 Materials and Methods
2.1 Selection of Cell Lines
In this study, two cell lines, MDA-MB-231 and MCF-7, were chosen as a model tumor type. Both cell lines are invasive ductal carcinomas and thus represent one of the most commonly encountered types of cancers. This also means that both cell lines are derived from the same tissue which helps limit the potential variations between tissue types. In addition, both cell lines have comparable doubling times and sizes. However, the MDA-MB-231 cell line has been identified as being far more aggressive and invasive than the MCF-7 cell line [22, 23, 24, 25, 26, 27, 28]. This key difference affects the makeup and proliferation of not only the cells themselves but also the ECM that surrounds them.
2.2 Synthesis of GNPs
The GNPs were synthesized via the reduction of HAuCl4 by sodium citrate, which is more commonly referred to as the Turkevitch’s method . By varying the amount of sodium citrate, this method can yield NPs of varying sizes. In summary, 300 µL of 1 % gold salt was added to 30 mL of doubled distilled water and brought to boil while stirring. The amount of reducing agent (1 % sodium citrate) added was 400 and 100 µL to produce 20 and 50 nm, respectively. The GNPs were characterized by transmission electron microscopy (TEM, H7000, Hitachi Corp. Tokyo, Japan), UV-spectroscopy (Lambda 40; PerkinElmer, Waltham, MA), and dynamic light scattering (DLS) using 90 Plus Particle Sizer Analyzer (Brookhaven Instruments Corp. New York, NY) to determine the size of the particles. The UV–visible peak wavelengths for 20 and 50 nm GNPs were 520 and 525 nm, respectively. According to TEM, the diameters of the NPs were 19.5 (±3.3) and 52.4 (±5.2). According to DLS measurements, the hydrodynamic diameters of the NPs were 24.2 (±0.6) and (52.6 ± 0.8). This information is given under supplementary information as well.
2.3 The CytoViva Imaging Technology
2.4 Growth of MCL Structures
To better model the tumor environment, cells were cultured on a semi-permeable membrane, as described by Minchinton et al. . The multi-layered cell culture develops into a thick mat of cells that acts as a barrier to the penetration of drugs and GNPs much in the same way as solid tumors and multicellular spheroids. They have been used to study the penetration of a variety of anticancer drugs as well as agents specifically targeted to hypoxic and nutrient deprived cells, where penetration is of the utmost importance . Previous work using MLCs demonstrated the development of hypoxia and necrosis in MLCs greater than 150-µm thick. The appearance of necrosis and hypoxia at 150 µm of tissue thickness presents an upward bound on the tissue sizes considered in this study [15, 18, 30, 31, 32, 33]. The effects of hypoxia and necrosis on the transport of GNPs were shown recently to be quite complex and will be pursued at the multilayer level in future studies .
Two breast cancer cell lines were used in this study: MCF-7 and MDA-MB-231. Cells were grown on the MLC insert in Dulbecco′s Modified Eagle′s Medium (LifeTechnologies Inc. Burlington, ON) with 10 % Fetal Bovine Serum (Sigma-Aldrich, Oakville, ON). Figure 3d shows an image of an unstained tissue cross section of MCF-7 cells. The ECM and cytoplasm of the cells within the tissue were stained with eosin for visualization (Fig. 3e). The darker areas in the image are the nuclei of cells. The nucleus will not be stained with eosin. The thickness of the tissue was controlled by the growth period. MLC incubation with NPs was conducted by hanging the MLCs 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 MLC to allow for GNPs that had penetrated the entire MLC structure to diffuse through. MLCs were examined and their thicknesses were measured. The thickness of an MLC was found by taking the average thickness over at least 10 slices.
2.5 Qualitative Analysis
To qualitatively measure the distribution of the GNPs as well as to provide a measure of MLC growth characteristics, MLC inserts were frozen in optimum cutting temperature (OCT) compound for sectioning. The frozen MLCs were then sectioned (Cryostat CM1900; Leica, Wetzlar, Germany) into 10–20-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 HSI dark-field microscope. By examining the images acquired by HSI, a qualitative examination of the layer-by-layer penetration was deduced.
3 Results and Discussion
3.1 Size Dependence of Gold Nanoparticle Uptake at Monolayer Level
3.2 Size Dependence of Gold Nanoparticle Transport at Multilayer Level Using MLC Model
Several studies have noted that the limiting effects of the ECM are tied to collagen content and the structural organization of the ECM itself [7, 8]. Although convection through the interstitial matrix is an important part of interstitial fluid and particle movement, it is limited by the hypertension found in the interstitium [11, 22, 39]. Many anticancer agents such as liposomes and gene vectors are large molecules ranging in size from 90 to 300 nm. Nanoparticle agents being developed also vary greatly in size ranging from 2 to 200 nm. Thus, diffusion through the interstitial matrix of the tumor presents a major barrier to the delivery of therapeutic agents and drugs.
In addition, cancer cells have been shown to disrupt and change the composition and makeup of the ECM. Invasive species in particular have been shown to break down the collagen that helps provide the ECM with its ability to limit the flow of foreign particles through tissue [25, 26]. The two cell lines used in this study are thus separated by the makeup and composition of their respective ECM. The measurements taken will establish what effect the ECM has on the transport of GNPs in tissue. According to Fig. 7c–d, NP penetration is better in deeper tissues in MDA-MB-231 in comparison to MCF-7. This could be due to the difference in ECM in these tumor tissues. One other factor to consider when discussing the penetration dynamics in solid tumors is the tortuosity of the path taken by particles through tumor tissue. Particles traversing or penetrating tissue are presented with cellular obstacles that force the particles on tortuous paths through the interstitial space [19, 31]. The tortuosity is not only difficult to measure but also will vary greatly between cell lines and also within a single tumor .
The authors would like to acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Foundation for Innovation (CFI), and Keenen Research Center for their support. The authors would also like to acknowledge Dr. Ian Tannock, Dr. Richard P. Hill, and Mrs. Carol Lee for their support.
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