Quantitative Imaging of Gold and Silver ... - ACS Publications

Letter pubs.acs.org/ac

Quantitative Imaging of Gold and Silver Nanoparticles in Single Eukaryotic Cells by Laser Ablation ICP-MS Daniela Drescher,*,†,‡ Charlotte Giesen,†,‡,§ Heike Traub,† Ulrich Panne,†,‡ Janina Kneipp,†,‡ and Norbert Jakubowski*,† †

BAM Federal Institute for Materials Research and Testing, Richard-Willstätter-Str. 11, 12489 Berlin, Germany Humboldt-Universität zu Berlin, Department of Chemistry, Brook-Taylor-Str. 2, 12489 Berlin, Germany

‡

S Supporting Information *

ABSTRACT: Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was utilized for spatially resolved bioimaging of the distribution of silver and gold nanoparticles in individual fibroblast cells upon different incubation experiments. High spatial resolution was achieved by optimization of scan speed, ablation frequency, and laser energy. Nanoparticles are visualized with respect to cellular substructures and are found to accumulate in the perinuclear region with increasing incubation time. On the basis of matrixmatched calibration, we developed a method for quantification of the number of metal nanoparticles at the single-cell level. The results provide insight into nanoparticle/cell interactions and have implications for the development of analytical methods in tissue diagnostics and therapeutics.

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information on the distribution of nanoparticles in a single cell is lost. In this Letter, we report high spatial resolution LA-ICP-MS to detect and quantify gold and silver nanoparticles in individual fibroblast cells. LA-ICP-MS requires suitable calibration procedures for accurate quantification, preferably using matrix-matched standards. This is difficult to achieve for complex biomatrixes such as cultured cells. Due to the lack of appropriate reference materials, several quantification methods based on matrix-matched laboratory standards have been proposed in the literature,22 e.g., spiked, frozen and embedded blood,23 tissue homogenate,24 and agarose gels.25 In tissue samples, detection limits for 232Th, 238U, and 195Pt were reported in the low μg kg−1 range.24,26 Here, we describe the quantification of gold and silver nanoparticles in single cultured cells based on a matrix-matched calibration using nitrocellulose membrane spiked with nanoparticle suspension.

he interaction of gold and silver nanoparticles with cells of animal origin has become a major field of research, driven by questions arising in different areas, ranging from therapeutic applications of gold nanoparticles1,2 over molecular plasmonics and analytical approaches3−5 to problems in nanotoxicology.6−8 Central to these problems is the quantification of nanoparticles in cells, which provides important information on, e.g., nanoparticle uptake under particular experimental conditions or the distribution of the nanoparticles in the cellular interior. Optical methods such as dark field microscopy,9 surfaceenhanced Raman scattering (SERS) imaging,4,10,11 and photothermal microscopy12,13 often rely on the plasmonic properties of the nanoparticles, which change during the interaction with the biological system.14 These methods show only limited use for quantification of nanoparticles in single cells. Electron and X-ray microscopy15 are in principle capable of providing precise information on nanoparticle distribution but require complicated preparation and are time-consuming. In the past few years, different analytical techniques have been developed that provide spatial information for bioimaging mainly to monitor elemental and molecular distributions in tissues.16 Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is a convenient alternative for elemental bioimaging in tissue sections, as it provides easy sample preparation, multielemental detection with high sensitivity, and high spatial resolution.17,18 The first applications of ICP-MS in solution using pneumatic nebulization were shown for single cell analysis.19 So far, such analyses have not been combined with the detection of metallic nanoparticles. Inorganic nanoparticles can be quantified by elemental analysis in particular by ICP-MS upon extraction or after digestion of the cell suspension20,21 but with the disadvantage that all spatial © 2012 American Chemical Society

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MATERIALS AND METHODS Silver and gold nanoparticles were synthesized by citrate reduction (Merck, Darmstadt, Germany) of silver nitrate and chloroauric acid (Sigma-Aldrich, Taufkirchen, Germany).27 Particle size distribution of citrate-stabilized nanoparticles was determined by dynamic light scattering and transmission electron microscopy giving a mean particle diameter of 25 ± 5 nm for gold nanoparticles (6.0·1014 particles/L) and 50 ± 15 nm for silver nanoparticles (1.2·1014 particles/L). Swiss albino mouse fibroblast cells (cell line 3T3; DSMZ, Braunschweig, Germany) were cultured in Dulbecco’s modified Received: September 12, 2012 Accepted: November 5, 2012 Published: November 5, 2012 9684

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Eagle medium (DMEM) supplemented with 10% fetal calf serum and 1% ZellShield (all from Biochrom AG, Berlin, Germany) and grown under standard conditions (37 °C and 5% CO2). For LA-ICP-MS experiments, 3T3 cells were grown on sterile coverslips (Thermo Fisher Scientific, Waltham, USA) in a 6-well plate and incubated with 1 mL of silver and gold nanoparticle suspension in standard cell culture medium. After an exposure time of 3 or 24 h, 3T3 cells were washed thoroughly with phosphate buffered saline (PBS), immediately fixed with 4% para-formaldehyde in PBS, and dehydrated in a graded series of ethanol for LA-ICP-MS analysis. LA-ICP-MS measurements of the isotopes 107Ag and 197Au were conducted with a NWR213 laser ablation system (ESI, Fremont, USA) coupled to an ICP sector field mass spectrometer (Element XR, Thermo Fisher Scientific, Bremen, Germany). For calibration, 0.5 μL of the gold or silver nanoparticle suspension with different concentrations were deposited on a Protran nitrocellulose (NC) membrane (poresize 0.45 μm, Schleicher and Schuell, Dassel, Germany) and dried. Each concentration was measured three times. NC membrane and fixed fibroblast cells were continuously ablated by line scans. Each raw data point is converted to a single pixel when the image is processed by Origin 8.5 (OriginLab Corporations, Northhampton, USA). The optimized working conditions of the LA-ICP-MS are compiled in Tables S1 and S2 (see Supporting Information).

Figure 1. LA-ICP-MS image of the 197Au+ intensity distribution (in cps) inside a single fibroblast cell and the corresponding bright field image (A). The fibroblast cell was incubated with gold nanoparticles in a particle concentration of 100 pM for 3 h. (B) Single line scans of the cell regions marked with an arrow in panel A. Every peak in the line scans represents nanoparticles and/or particle aggregates. Parameters: laser spot size, 4 μm; line distance, 6 μm; scan speed, 5 μm/s; repetition rate, 10 Hz; pixel size, 1 × 6 μm; fluence, 0.8 J/cm2.

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RESULTS AND DISCUSSION Imaging of Nanoparticles in Fibroblast Cells. Fibroblast cells were incubated with gold and silver nanoparticle suspensions and grown as a monolayer on sterile coverslips for ICP-MS investigation, fixed with formaldehyde, and dried prior to ablation. To minimize fractionation effects during LAICP-MS analysis, laser energy, repetition rate, spot diameter, and scan speed were optimized so that the material was completely ablated during every line scan. In order to obtain a high resolution image with a spot size in scan direction smaller than the laser focus point diameter, here we applied a laser ablation scan mode that is based on the overlap of single ablation points. This procedure requires (i) the laser energy to be high enough for complete ablation and for minimum background signal and (ii) the repetition rate high enough relative to scan speed, so that the laser spots on the surface of the sample are overlapping and the signal of the sample is generated by the difference in ablated area. To investigate the particle uptake and distribution with respect to particle type, concentration, and incubation time, we analyzed individual fibroblast cells. It is known that most gold and silver nanoparticles are internalized through an endocytotic pathway and are localized in vesicles inside the cell.11,14,28 In Figure 1, plots of 197Au+ signal intensity as a function of position show the distribution of Au nanoparticles in a single fibroblast cell (A) and in three selected line scans (B) of the cytoplasmic region and the cell nucleus of the same cell. For these investigations, a laser spot size of 4 μm (line distance of 6 μm), a scan speed of 5 μm/s, and a repetition rate of 10 Hz were applied. The image pixel size of 1 μm in scanning (x)direction and 6 μm in y-direction is determined by the measurement time per data point and the line distance, respectively. This high spatial resolution enables the detection of nanoparticle agglomerates in the cell. Every peak in the line scans (Figure 1B) represents either single metal nanoparticles or particle aggregates that are localized inside endosomes or

lysosomes.6,28 The line scans of the cytosol are characterized by spots with high intensities, indicating elevated gold nanoparticle concentration (Figure 1B). The 2D map in Figure 1A indicates a high quantity of gold nanoparticles in the perinuclear region, which is in accordance with previous results from various ultra/-microscopic experiments.11 Since bare nanoparticles above 20 nm in diameter cannot enter the nucleus,28,29 only low signal intensity is found in the region of the nucleus (Figure 1, red scan line) that originates from Au nanoparticles in the cytosol volume above or below the nucleus. This example illustrates that the LA-ICP-MS approach enables one to localize gold nanoparticles and their aggregates within the substructures of single fibroblast cells and therefore can be used to investigate nanoparticle uptake and intracellular distribution. Figure 2 displays an overlay of a color encoded 107Ag+ local intensity map with bright field micrographs for three nanoparticle concentrations and two incubation times. In order to increase the sensitivity in these experiments, a laser spot size of 8 μm was used, and a larger area from the slide, comprising several fibroblast cells, was investigated. As clearly visible in the maps (Figure 2), the resulting resolution is sufficient to differentiate between the cell nuclei, cytosol, and background. As observed for gold nanoparticles, no significant contribution to the 107Ag+ intensity was measured in the nucleus region (compared to Figure 1A). The uptake rate can be compared for the different experimental conditions. As visible in Figure 2A−C, the intensity of 107Ag+ and thus the concentration of silver nanoparticles inside the cells increases with an increase in nanoparticle concentration (0.2−20 pM nanoparticle concentration). Likewise, at 20 pM concentration, a longer incubation 9685

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Figure 2. Bright field images of fixed 3T3 fibroblast cells superimposed with LA-ICP-MS images of the 107Ag+ intensity distribution (in cps). Cells were incubated with silver nanoparticles in a concentration of 0.2 to 20 pM for 3 h (D) or 24 h (A−C). Scale bars represent 25 μm. Parameters: laser spot size, 8 μm; scan speed, 8 μm/s; repetition rate, 5 Hz; pixel size, 1.2 × 8 μm; fluence, 1.5 J/cm2.

time, increased from 3 to 24 h, also yields higher 107Ag+ intensity (compare Figure 2C,D). In addition to differences in intensity, also distribution varies after different incubation times. While particles are found to be evenly distributed in the cytosol after 3 h of incubation (Figure 2D), they accumulate in the perinuclear region after longer exposure (Figure 2C). These observations are in accordance with the results of other studies on the distribution of silver nanoparticles in cells.11,30 Quantification of Gold and Silver Nanoparticles in Individual Cells. LA-ICP-MS also features an exceptional accuracy and ease of calibration in element quantification. Most calibration methods require the use of an internal standard (e.g., 13C isotope standard) to account for matrix dependence of the ablation process, variations in mass ablated, differences in transport efficiency, and instrumental drift.31 A recent study reported differences in the transport mechanisms of carbon (gaseous phase) and analytes (particulate phases), that cause difficulties in the use of 13C as an internal standard for quantification in biological applications using nonmatrixmatched calibration standards.32 Previously, we showed the use of iodine as an internal standard.18 However, as iodination affects the incubation of gold and silver nanoparticles in the cells, it cannot be used for quantification of these nanomaterials. Thus, we must rely on an absolute approach, which is valid in this work, since the sample is fully ablated. As most of the aerosol transported to the plasma consists of carbon-rich particles, we have selected nitrocellulose membrane for matrixmatching calibration. This material has been successfully used for calibration in proteomics due to its excellent absorption and high cleanliness.33 In the calibration experiment, a series of dried gold and silver nanoparticle droplets on a nitrocellulose membrane were completely ablated and analyzed by ICP-MS. Figure S1 of the Supporting Information displays the calibration graphs, which were linear within the calibration range. The limit of detection and the limit of quantification for

silver nanoparticles with a mean diameter of 50 nm were 20 and 60 particles and for gold nanoparticles with a diameter of 25 nm were 190 and 550 particles, respectively. Single fibroblast cells, incubated with silver or gold nanoparticles, were ablated, and the number of particles per cell was estimated on the basis of these calibration curves (Figures S1 and S2, Supporting Information). The number of nanoparticles is given as mean value of 6 to 10 cells for each incubation condition (Figure 3A,B). Due to cell variability in size, morphology, and nanoparticle uptake, differences from one cell to the other occur, that is reflected by the standard deviation bars. As discussed above (Figure 2), silver nanoparticle uptake strongly depends on particle concentration and incubation time. Cells incubated with silver nanoparticles in a concentration of 0.2 to 2 pM for 24 h contain 380 and 2500 particles per cell, respectively (Figure 3A). While these concentrations are known to be nontoxic to the fibroblast cells, a higher nanoparticle concentration of 20 pM, that proves to be cytotoxic in the XTT test (cell proliferation/viability assay), yields 15 000−57 000 particles per cell, depending on the exposure time. A strong concentration dependency of particle uptake is found for the gold nanoparticles (Figure 3B), with 2400, 19 000, and 160 000 particles per cell after a 24 h incubation for particle concentrations of 5, 10, and 100 pM, respectively. This corresponds to a gold content of 0.3 to 19.5 pg per cell, which is in accordance with ICP-MS studies on eukaryotic cells after digestion and extraction of the nanoparticle−cell suspension.34,35 The quantification of Au nanoparticles results in much higher numbers at similar nanoparticle concentration in the cell culture medium due to smaller particle size and larger uptake efficiency into the cells compared to silver nanoparticles. Considering all aspects, the results of the present publication are in good agreement with results of experiments utilizing other analytical techniques. 9686

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Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (D.D.); norbert. [email protected] (N.J.). Present Address §

University of Zurich, Institute of Molecular Life Sciences, Winterthurerstr. 190, 8057 Zurich, Switzerland. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS D.D. and J.K. acknowledge funding from ERC Grant 259432 (MULTIBIOPHOT).

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Figure 3. Quantification of silver (A) and gold (B) nanoparticles inside single fibroblast cells by LA-ICP-MS based on matrix-matched calibration data (Figure S1, Supporting Information). Cells were exposed to nanoparticles in different concentrations for 3 and 24 h. Parameters: laser spot size, 8 μm; scan speed, 8 μm/s; repetition rate, 5 Hz; fluence, 1.5 J/cm2.

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CONCLUSION It was demonstrated that high spatial resolution LA-ICP-MS can be utilized in bioimaging to determine the intracellular nanoparticle distribution in cellular substructures of individual cells. Specifically, it can be applied to quantify the number of metal nanoparticles at the single-cell level. Subcellular mapping, enabling differentiation of nanoparticles in the cytosol from those in the cell nucleus region, was achieved by improving the spatial resolution through optimization of the ablation/ scanning parameters. Single 3T3 cells, incubated with silver or gold nanoparticles, show a strong dependency of particle uptake on concentration and incubation conditions. Quantification of gold and silver nanoparticles in single cells is possible based on a matrix-matched calibration using nitrocellulose membranes doped with nanoparticle suspension. Our results demonstrate the potential of LA-ICP-MS for nanotoxicity investigations, nanobioanalytics, and quantitative elemental microscopy.

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REFERENCES

ASSOCIATED CONTENT

S Supporting Information *

Additional information on experimental methods. Table S1: NWR213 operating parameters. Table S2: Element XR operating parameters. Figure S1: Calibration graphs of silver and gold nanoparticle suspensions. Figure S2: LA-ICP-MS image of the 107Ag+ and 197Au+ intensity distribution and a line scan of the lowest nanoparticle concentration used for matrixmatched calibration. This material is available free of charge via the Internet at http://pubs.acs.org. 9687

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