Assessment of Metal Nanoparticle Agglomeration,Uptake,and Interaction Using High-Illuminating System

Abstract

In the present study, an ultrahigh-resolution system was applied as a simple and convenient technique to characterize the extent of metal nanoparticle agglomeration in solution and to visualize nanoparticle agglomeration, uptake, and surface interaction in three cell phenotypes under normal culture conditions. The experimental results demonstrated that silver (25, 80, 130 nm); aluminum (80 nm); and manganese (40 nm) particles and agglomerates were effectively internalized by rat liver cells (BRL 3A), rat alveolar macrophages (MACs), and rat neuroendocrine cells (PC-12). Individual and agglomerated nanoparticles were observed within the cells and agglomerates were observed on the cell surface membranes. The particles were initially dispersed in aqueous or physiological balanced salt solutions and agglomeration was observed using the Ultra Resolution Imaging (URI) system. Different methods, such as sonication and addition of surfactant (0.1% sodium dodecyl sulfate [SDS]) reduced agglomeration. Due to effects of SDS itself on cell viability, the surfactant could not be directly applied during cell exposure. Therefore, following addition of 0.1% SDS, the particles were washed twice with ultrapure water, which reduced agglomeration even further. Reducing the agglomeration of the nanoparticles is important for studying their uptake and in applications that benefit from individual nanoparticles such as diagnostics. In summary, this study demonstrates a simple technique to characterize the extent of nanoparticle agglomeration in solution and visualize nanoparticle (40 nm and larger) uptake and interaction with cells. Additionally, an example application of nanoparticle labeling onto the surface and neurite extensions of murine neuroblastoma cells (N2A) is presented as a potential imaging tool.

Nanoparticles, defined as having at least one dimension of 100 nanometers or less, are being used to create novel materials that possess unique physicochemical properties (Hood 2004). Some of the intended applications for nanotechnology include chemical and biological detectors for homeland defense, terahertz processors, and lightweight composite materials, which are many times stronger than steel (Freund et al. 2005). Biocompatible nanoparticles can be used in various biomedical applications; for example, bone cement may be loaded with nanosilver particles (5 to 50 nm) which act as an antibacterial agent to multiresistant bacteria in artificial joints (Alt et al. 2004). In addition, nanoparticles may be used in biosensors (Lee and El-Sayed 2006), medical imaging (Corot et al. 2006), and tissue engineering (Hill and Shear 2006).

The rapidly developing field of nanotechnology will result in exposure of nanoparticles to humans via several routes (e.g., inhalation, ingestion, skin uptake, injection, etc.), validating the need for toxicity studies to determine any deleterious effects of these nanoparticles on living cells. An important step in carrying out toxicity studies of nanoparticles is studying their uptake and interaction with cells. As materials reach the nanoscale, the surface area per unit mass increases, therefore increasing their potential for surface chemistry effects during biological interaction (Oberdoster et al. 2005). Current studies have shown that nanoparticles can penetrate cells and translocate from their route of exposure to other vital organs (Hoet et al. 2004). One study found that in nanoparticle uptake, the cell membrane invaginates to form a vesicle around the particle (Gorelik et al. 2002). In our laboratory, we found that silver nanomaterials induced oxidative stress in cultured BRL 3A rat liver cells (Hussain et al. 2005) and were more toxic to germ line stem cells (Braydich-Stolle et al. 2005) when compared to other stable cultured cells.

There are many factors that must be taken into account when evaluating the potential toxicity of nanoparticles, specifically, shape, surface area, surface chemistry, dimension, and agglomeration in solution (Soto et al. 2004). The process of agglomeration may occur during nanoparticles synthesis or once the nanoparticles are introduced into various solutions where electrostatic interactions or chemical bonding may lead to aggregates. Due to the nanosize and the nature of agglomeration in these particles, no simple standard method to characterize uptake into the cells is currently available.

The following study evaluates the uptake of nanoparticles through the use of a ultrahigh-resolution system (Aetos Technologies, Auburn, AL), which uses an advanced illumination system attached to a standard research grade inverted microscope. Because the introduction of high numerical aperture objectives, the accepted limit of resolution for light microscopes has been approximately 250 nm (Foster 2004); however, the Ultra Resolution Imaging (URI) system provides an image resolution of less than 150 nm (Vodyanoy 2005; Foster 2004). In the present study, silver (25, 80, 130 nm), aluminum (80 nm), and manganese (40 nm) were examined in three different cell lines with reference to application of nanoparticle agglomeration in aqueous and physiological solution and this effect on cellular uptake and interaction. Additionally, a silver nanoparticle coated with polysaccharide (organically functionalized for increased dispersion) was examined as a biolabel for decoration of the cellular surface and neurite extensions.

MATERIALS AND METHODS

CytoViva150 Ultrahigh Resolution Imaging (URI) System

The CytoViva150 Ultra Resolution Imaging (URI) system is an illumination system with a cardioid annular condenser (annular A-condenser), replacing the regular bright-field condenser (circular C-condenser) on a standard research transmission optical microscope (Aetos Technologies). The illumination system is connected by a liquid light guide with a light source (EXFO120; Photonic Solution), which focuses light onto the annular entrance slit of the annular A-condenser. The annular A-condenser produces a narrower diffraction pattern than the circular C-condenser, resulting in resolution of better than 90 nm (Vainrub et al. 2006).

Cell Lines and Chemicals

All cell line used in this study were obtained from American Type Culture Collection (Manassas, VA) with the exception of murine neuroblastoma (N2A) cells, which were a kind gift from Dr. David Cool of the Wright State University, Dayton, OH. BRL 3A (ATCC, CRL-1442) immortalized rat liver cells were used between passages 10 and 20. The PC-12 cell line, which was derived from a rat pheochromocytoma (ATCC, CRL-1721) was used between passages 10 and 15. Rat alveolar macrophages (ATCC, NR8383), abbreviated MACs, were used between passages 10 and 20. Culture media, penicillin-streptomycin, and other chemicals were purchased from Sigma Chemical (St. Louis, MO).

Cell Culture

BRL 3A cells were grown in Ham’s nutrient mixture F-12 medium, pH 7.25, with 5% fetal bovine serum (FBS). N2A cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium with 10% FBS. PC-12 cells were grown in RPMI-1640 medium supplemented with heat inactivated 5% FBS and 10% horse serum. MACs were maintained in modified F-12K medium supplemented with 20% FBS and 2 mM l-glutamine. All media contained 1% penicillin-streptomycin as an antibiotic. Cells were grown on chamber slides before exposure with the nanoparticles. The cells were maintained in a 5% CO₂ incubator at 37°C and 100% humidity.

Nanoparticles

Silver (Ag; 25, 80, 130 nm) and aluminum (Al; 80 nm) nanoparticles were a kind gift from Dr. Karl Martin at Nanotechnologies, Austin, TX. Manganese (Mn; 40 nm) nanoparticles were a kind gift from Dr. Gunter Oberdorster, University of Rochester School of Medicine and Dentistry, Rochester, NY, and his laboratory associates. Silver nanoparticles approximately 25 to 30 nm in size with a polysaccharide/organic coating (Ag25–30Disp) were a kind gift from Dr. Dan Goia at Clarkson University, NY.

Preparation of Dosing Solution for Exposure

The silver and aluminum nanoparticles were dispersed in sterile ultrapure water at a concentration of 10 mg/ml as a stock solution. The polysaccharide/organic-coated silver nanoparticles were prepared at a stock concentration of 1 mg/ml. The final dosing concentrations of 5, 10, and 25 μg/ml were prepared in cultured medium from the stock solutions. Because of the instability of suspension and quick agglomeration in water, the manganese nanoparticles were dispersed in a physiological saline solution (pH 7.4) at concentrations of 5, 10, and 25 μg/ml.

Due to excessive agglomeration, some of the Ag nanoparticles were prepared in a 0.1% sodium dodecyl sulfate (SDS) solution, an anionic surfactant. The nanoparticles were then washed twice to remove excess surfactant, then filtered through a centrifugation filter (10 μm) with ultrapure water and resuspended in ultrapure water by vortex mixing. Immediately before exposure, the nanoparticle solutions were sonicated for 10 s to further reduce nanoparticle agglomeration, though homogenous dispersion of the nanoparticles used in these studies was not achieved.

Method for Visualizing Nanoparticles in Solution

Aliquots of nanoparticles (10 μl) dispersed in aqueous or physiological solutions (1 mg/ml) were placed on glass slides and covered with coverslips. The coverslips were sealed in place by applying regular clear nail polish to the edges. The slides were inverted and a few drops of immersion oil were applied to the back of the slides. The slides were then secured on the Olympus IX71 Microscope platform that was coupled with the URI System. The URI system lens was adjusted so it just touched the oil on each slide. The light was focused and centered in the microscope’s 10× air lens. One or two drops of immersion oil were placed on the 60× oil lens, and the objective was brought up until the oil made contact with the slide and images were finely focused. QCapture Pro Imaging Software was used to capture and store images.

Exposure Protocol

The cells were grown on dual chamber slides under physiological conditions for 48 h. When the cells were 70% confluent on the slides, they were treated for 24 h with a range of concentrations (5 to 25 μg/ml) of Ag (25, 80, 130 nm), Al (80 nm), and Mn (40 nm) particles. The organically polysaccharide/organic-coated Ag nanoparticles were applied only at a concentration of 100 μg/ml. After 24 h of exposure, the excess medium was removed and the cells were washed twice with physiological saline solution in order to remove unbound nanoparticles. The chambers were removed with a chamber slide remover and coverslips were carefully placed on the slides. The coverslips were sealed in place using regular clear nail polish. The slide was then observed as described in the preceding section.

Measurement of Particle Sizes Using QCapture Pro Imaging Software

To calibrate each objective, a line was defined across the horizontal and vertical axes of a known reference object (supplied by Aetos Technologies), and the units were specified in x and y directions. QCapture Pro software could then be used to relate this distance to pixels by dividing the number of pixels under the defining line by the specified units.

To add a scale or measure bar to an image, the objective used in acquiring the image (e.g., 60×) was selected from the computer software drop-down menu in order to apply the correct calibrated measurement per pixel. Once the scale bar is burned onto the image, it is permanently attached to the image and any future modifications to the image do not affect the accuracy of the measurement.

To measure distances, the image was magnified in order to get a more accurate view of the end points of each particle agglomerate, then a line was drawn from one end to the other across the longest axis. Each pixel at 60× represented roughly 0.0776 microns in both the x and y directions (pixels are square). Therefore, measurements smaller than this value cannot be made, and measurements are all approximate. It is important to remember that the number of pixels of an image changes when saved under a different file extension, and therefore, measure bars and distance measurements are only accurate scales when applied to original captured images.

RESULTS

The first set of experiments was conducted to observe particle dispersion in aqueous solutions. The major issue encountered was non-homogenous dispersion, shown in Figure 1, which includes images of 130-nm and 25-nm Ag particles in ultrapure water (1 mg/ml). Agglomerate sizes were measured in captured images as described in the methods section. The extent of agglomeration of the particles in solution changed based on the method of preparation. When the 130-nm Ag particle solution was vortexed, it showed a large amount of agglomeration as indicated in Figure 1A . Agglomerates measured ranged in size from approximately 200 nm to over 16 microns. Next, a surfactant was added to the solutions to test its ability to aid in the dispersion of the nanoparticles. Figure 1B shows sonicated 130-nm Ag in 0.1% SDS solution and less agglomeration is present compared to Ag dispersed in water only. The size of the agglomerates was as large as 27 microns, and most of the particles were between 150 and 250 nm. When the solutions containing 0.1% SDS were washed twice with ultrapure water before being added to the slide, some agglomerates, up to 14.5 microns across the longest axis, were present. However, many more small agglomerates, which were barely visible, were also present and were approximately 150 nm in size (Figure 1C ).

Figure 1D represents extensive agglomeration of 25-nm Ag particles, which were only vortexed. This solution appears to contain large agglomerates approximately 12 microns in diameter and several smaller agglomerates approximately 200 to 300 nm in diameter, demonstrating a lower degree of agglomeration compared to the vortexed 130-nm Ag solution in Figure 1A . Figure 1E indicates that 25-nm Ag particles suspended in deionized water with 0.1% surfactant agglomerated up to 4.5 microns in size and some agglomerates measured approximately 150 nm. The same solution containing 25-nm Ag, which was washed twice with ultrapure water appeared to contain a very slight reduction of agglomeration compared to the solution that was not washed. Several agglomerates are visible at sizes of approximately 78 nm. Due to software limitations (each pixel equals 0.0776 microns), agglomerates or particles smaller than 78 nm cannot be measured.

The next set of experiments investigated whether nanoparticles and agglomerates are internalized and how they interact with the cells after 24 h of exposure. Figure 2 demonstrates the uptake of nanoparticles by three different cell lines. It is important to note that small circular dots are seen in the cytoplasm of untreated BRL cells (Figure 2A and D ). These dots are likely to represent cell line–dependent granules or organelles, which naturally provide contrast with the URI system. All particle solutions used for BRL cell exposure were prepared with 0.1% SDS, which was washed away by two rounds of centrifugations. Figure 2B indicates several agglomerates of 80-nm Ag particles on the membrane and possibly in the cytoplasm of a BRL cell. Figure 2C shows 80-nm Ag agglomerates on the cell membrane periphery as well as smaller agglomerates in the cytoplasm of the BRL cells. These particles have permeated the cell membrane, but not the nuclear membrane. Figure 2E illustrates aggregates of 25-nm Ag nanoparticles (10 μg/ml) in the cytoplasm of the BRL cells. The nanoparticles are distinguished from organelle membrane reflections observed in the control by their larger size and greater intensity. In Figure 2F , 25-nm Ag particles at a concentration of 10 μg/ml outside the BRL nuclear membrane are indicated. Figure 2G illustrates control PC-12 cells. Figure 2H shows PC-12 cells that were incubated with 25 μg/ml of sonicated 40-nm Mn. Some particles are internalized into the cytoplasm of the cells, but did not appear to enter the nuclear region. Again, highly agglomerated particles have not been internalized. This same phenomenon is shown in Figure 2I . Figure 2J illustrates untreated MACs and Figure 2K demonstrates aggregated Al particles on the membrane of cells treated with 25 μg/ml of sonicated 80-nm particles. Aggregated particles also appear to be located just inside the membrane. Figure 2L shows that the alveolar MACs have internalized large aggregates of particles.

The use of organically functionalized silver nanoparticles ranging in size from 25 to 30 nm show that they can beautifully orient onto the neurites of N2A cells (Figure 3C ). Using the URI system, the binding of the biocompatible Ag nanoparticles was shown with a high brightness after 24 h of incubation with a 100-μg/ml solution of the nanoparticles in cell culture medium while rinsing did not easily dislodge the nanoparticles. A comparison was made with control cells (Figure 3A ) without nanoparticle treatment and nano-sized silver (Figure 3B ) without an organic coating, which does not show the binding to neurites, but rather reduces neurite extension and encourages rounding.

DISCUSSION

Increases in the manufacture and use of man-made nanoparticles will likely increase the amount and duration of nanomaterials exposure for both humans and animals. Many studies have evaluated the toxicity of engineered nanoparticles both in vitro and in vivo (Hussain et al. 2005; Lam et al. 2004). However, it is important that more detailed investigations into the mechanisms that introduce nanomaterials into cells be performed in order to fully understand the toxicity of nanoparticles. Scanning electron microscopy and atomic force microscopy have been used to image cells, but their application in the field of biological sciences is limited (Gorelik et al. 2002). Previous studies utilizing transmission electron microscopy have shown aggregation and internalization of manufactured nanoparticles including silver (Soto et al. 2005). However, there is no simple method to visualize the extent of agglomeration and uptake of nanoparticles.

This study using the URI system attached to a common laboratory microscope has demonstrated that the uptake of various nanomaterials into the cytoplasm of BRL, PC-12, and MAC cells can be visualized while the cells are living. Further, nanomaterial interaction with living cells can be observed and studied for kinetics and subsequent response signature. Previous inhalation toxicity studies in vivo have shown that the deposition of nanoparticles is size specific where single ultrafine particles are inhaled rather than aggregate, which contributes to their effect on toxicity (Warheit 2004). Another inhalation study by Brown et al. (2001) demonstrated that the increased surface area and reactivity of ultrafine particles has an effect on proinflammatory responses. A simulation of particle uptake by Welzel et al. (2004) concluded that the transfection of cells with nanoparticles appears to be independent of the chemical nature of the nanoparticle, but rather depends mostly on the particle size. This experiment demonstrated, as do our microscopic evaluations, that many of the larger aggregates were not internalized by the cells in comparison to individual particles.

To reduce the aggregation of particles around the cells, the particles were treated with a 0.1% solution of SDS. It has been shown that a high concentration of SDS and high ultrasonic pulse power can produce a stable solution of gold nanoparticles (Park, Atobe, and Fuchigami 2005). Because of the concomitent toxicity of a high percentage of SDS, a low concentration (0.1%) was used, followed by two washes with deionized water. The addition of SDS to the stock solutions of nanoparticles seemed to slightly decrease the amount of aggregation in silver particles and thereby increase the amount of internalization of these particles within the cells. It is still debatable whether nanoparticle agglomeration contributes to nanotoxicity. This study shows that agglomeration may have affected the uptake of nanomaterials into the cells, which is shown in Figures 1 and 2. However, at this resolution, the exact location of the nanoparticles inside the cells is still not known.

Overall, the ultrahigh resolution system is an effective means to quickly observe solutions of nanoparticle agglomerates and their uptake into cells with a record of high-resolution, high-contrast images. Although a great deal of unanswered questions remain in regards to nanotoxicity, this procedure shows an effective means to visualize particle agglomerates inside the cells or on the cell surface. It is a well-known fact that agglomeration occurs in the majority of man-made nanomaterials with high surface activity and may increase over time. Therefore, understanding of how nanoparticles agglomerate in solution and are incorporated into or onto cells is critical to accelerate the understanding of bioeffects and toxicological response with limited budgets and time on time-specific events of these exposures. Additionally, the development of small, dispersible, and bio-compatible nonfluorescent biological labels can be rapidly visualized to demonstrate their feasibility as imaging probes or in many other emerging technologies. Future studies can expect to profit from the simplistic method of in vitro cell culture analysis with fluorescent techniques, which may be better suited to answer questions regarding localization and possible transfer of the technology to in vivo situations. In conjuction with ultrahigh-resolution ligh microscopy, nanoparticle uptake by electron microscopy at selected time points will provide more detailed results.