Monitoring the Uptake and Redistribution of Metal Nanoparticles

Aug 9, 2010 - Narayana M. S. Sirimuthu, Christopher D. Syme, and Jonathan M. Cooper* .... (23) Tsang, J. C.; Kirtley, J. R.; Bradley, J. A. Phys. Rev...
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Anal. Chem. 2010, 82, 7369–7373

Monitoring the Uptake and Redistribution of Metal Nanoparticles during Cell Culture Using Surface-Enhanced Raman Scattering Spectroscopy Narayana M. S. Sirimuthu, Christopher D. Syme, and Jonathan M. Cooper* Department of Electronics and Electrical Engineering, The Bioelectronics Research Centre, University of Glasgow, Glasgow, G12 8LT, Scotland, U.K. We describe the uptake of silver nanoparticles by CHO (Chinese hamster ovary) cells and their subsequent fate as a result of cell division during culture, as monitored by surface-enhanced Raman scattering (SERS) spectroscopy. Mapping of populations of cells containing both labeled and native nanoparticles by SERS spectroscopy imaging provided a quantitative method by which the number of intracellular nanoparticles could be monitored. Initially, for a given amount of nanoparticles, the relationship between the number taken up into the cell and the time of incubation was explored. Subsequently, the redistribution of intracellular nanoparticles upon multiple rounds of cell division was investigated. Intracellular SERS signatures remained detectable in the cells for up to four generations, although the abundance and intensity of the signals declined rapidly as nanoparticles were shared with daughter cells. The intensity of the SERS signal was dependent both on stability of the label and their abundance (nanoparticle aggregation increases the extent of the SERS enhancement). The data show that while the labeled nanoparticles remain stable for prolonged periods, during cell division, the changes in signal could be attributed both to a decrease in abundance and distribution (and hence aggregation). Nanoscale silver and gold particles exhibit distinct chemical and physical properties, when compared to their bulk metals, leading to a variety of distinct applications in biology,1-5 electronics,6 and chemistry.7,8 In biology, there has been a significant increase in the diversity of applications of such nanoparticles, including their use as labels in electron microscopy,9 target * Corresponding author. Tel: +44(0)1413304931. Fax: +44(0)1413304907. E-mail: [email protected]. (1) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547–1562. (2) Sinha, R.; Kim, G. J.; Nie, S. M.; Shin, D. M. Mol. Cancer Ther. 2006, 5, 1909–1917. (3) Daniel, M. C.; Astruc, D. Chem. Rev. 2004, 104, 293–346. (4) Murphy, C. J.; Gole, A. M.; Hunyadi, S. E.; Stone, J. W.; Sisco, P. N.; Alkilany, A.; Kinard, B. E.; Hankins, P. Chem. Commun. 2008, 544–557. (5) Durr, N. J.; Larson, T.; Smith, D. K.; Korgel, B. A.; Sokolov, K.; Ben-Yakar, A. Nano Lett. 2007, 7, 941–945. (6) Sargent, E. H. Adv. Mater. 2005, 17, 515–522. (7) Kong, T. S. A.; Yu, K. M. K.; Tsang, S. C. J. Nanosci. Nanotechnol. 2006, 6, 1167–1172. (8) Guo, Z. F.; Ma, R. X.; Li, G. J. Chem. Eng. J. 2006, 119, 55–59. (9) Baschong, W.; Wrigley, N. G. J. Electron Microsc. Tech. 1990, 14, 313– 323. 10.1021/ac101480t  2010 American Chemical Society Published on Web 08/09/2010

selected structures for therapy,10,11 and vehicles for drug delivery.12,13 Both silver and gold nanoparticles have been used as substrates for intracellular optical studies.14-17 Raman spectroscopy is a nondestructive and noninvasive technique which requires minimal sample preparation and can often be applied to label-free analyses. It provides molecular information through the measurement of specific characteristic vibrational frequencies present within a sample. The narrow spectral bands compared to fluorescence signatures make it a versatile analytical technique for the characterization of the composition of complex biochemical mixtures, such as those found in cells.18 In general, the applicability of Raman spectroscopy is limited due to its inherent weakness, since only a small proportion of light is inelastically scattered (approximately one in a million photons). Even with advances in detector technologies, acquisition times for Raman spectroscopy often remain prohibitively long, especially when spatial mapping of biomaterials is being performed. However, the efficiency of Raman scattering can be enhanced either by surface enhancement (SERS) or by surface enhancement coupled with the resonance Raman effect (SERRS), where the light interacts with a nanostructured metal and the analyte being measured. As a consequence, both SERS and SERRS spectroscopies have been reported as being able to increase the magnitude of the signal by many orders of magnitude.19,20 As stated, SERS and SERRS spectroscopies both rely upon a surface enhancement effect that involves the analyte interacting with a surface plasmon generated at a suitable metal. A variety of (10) de la Fuente, J. M.; Berry, C. C. Bioconjugate Chem. 2005, 16, 1176–1180. (11) Feldherr, C. M.; Kallenbach, E.; Schultz, N. J. Cell Biol. 1984, 99, 2216– 2222. (12) Han, G.; You, C. C.; Kim, B. J.; Turingan, R. S.; Forbes, N. S.; Martin, C. T.; Rotello, V. M. Angew. Chem., Int. Ed. 2006, 45, 3165–3169. (13) Sandhu, K. K.; McIntosh, C. M.; Simard, J. M.; Smith, S. W.; Rotello, V. M. Bioconjugate Chem. 2002, 13, 3–6. (14) Zhang, X. L.; Yin, H. B.; Cooper, J. M.; Haswell, S. J. Anal. Bioanal. Chem. 2008, 390, 833–840. (15) Scaffidi, J. P.; Gregas, M. K.; Seewaldt, V.; Vo-Dinh, T. Anal. Bioanal. Chem. 2009, 393, 1135–1141. (16) Chourpa, I.; Morjani, H.; Riou, J. F.; Manfait, M. FEBS Lett. 1996, 397, 61–64. (17) Kyriacou, S. V.; Brownlow, W. J.; Xu, X. H. N. Biochemistry 2004, 43, 140– 147. (18) Faulds, K.; Jarvis, R.; Smith, W. E.; Graham, D.; Goodacre, R. Analyst 2008, 133, 1505–1512. (19) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R.; Feld, M. S. Phys. Rev. Lett. 1997, 78, 1667–1670. (20) Qian, X. M.; Nie, S. M. Chem. Soc. Rev. 2008, 37, 912–920.

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such substrates have been reported, including a number of different metals in various morphologies, including colloidal suspensions,21,22 gratings,23 island films,24 nanolithographically structured geometries,25 and films deposited on paper substrates.26 The most common substrates used in intracellular measurements are gold and silver colloids (often referred to as nanoparticles) since they can be easily prepared, characterized, and readily introduced into cells. Silver colloid was used in this study as it gives a greater enhancement (due in part to the higher conductivity of the metal)27 and consequently could be used at concentrations that were not toxic to the cells. It is known that nanoparticles can be easily introduced into cells in a variety of different ways, with passive uptake of native (unmodified) particles being the most straightforward and most commonly used method.28-30 The toxicity of metal nanoparticles has also previously been studied for different cell lines31,32 exploring the fate (including using SERS33) of both the nanoparticles34 and of labels, attached to the nanoparticles35 over short time periods (e.g., typically hours after uptake into the cell). The size, shape, and concentration dependence of nanoparticle uptake has been previously explored using UV spectroscopy and inductively coupled plasma atomic emission spectroscopy (ICP-AES).36 There has been considerably less work that has sought to explore the fate of the cells beyond a single generation, investigating the distribution of the particles after successive rounds of cell division. This is of particular interest to those interested in the use of labeled nanoparticles over extended periods, to probe changes in the cell biochemistry (including for example, pH).37 In this study, we now explore the fate of such nanoparticles during cell culture and division, to understand how nanoparticles redistribute themselves in the cell over extended periods of time. In order to measure the uptake and the distribution of the nanoparticles, we use the SERS signal from both 4-mercaptobenzoic acid (4-MBA) labeled silver and from native (bare) silver colloid. (21) Bell, S. E. J.; Sirimuthu, N. M. S. Chem. Soc. Rev. 2008, 37, 1012–1024. (22) Bell, S. E. J.; Sirimuthu, N. M. S. J. Am. Chem. Soc. 2006, 128, 15580– 15581. (23) Tsang, J. C.; Kirtley, J. R.; Bradley, J. A. Phys. Rev. Lett. 1979, 43, 772– 775. (24) Li, P. W.; Zhang, J.; Zhang, L.; Mo, Y. J. Vib. Spectrosc. 2009, 49, 2–6. (25) Clark, A. W.; Glidle, A.; Cumming, D. R. S.; Cooper, J. M. Appl. Phys. Lett. 2008, 93. (26) Tran, C. D. Anal. Chem. 1984, 56, 824–826. (27) Sheng, R. S.; Zhu, L.; Morris, M. D. Anal. Chem. 1986, 58, 1116–1119. (28) Kneipp, J.; Kneipp, H.; McLaughlin, M.; Brown, D.; Kneipp, K. Nano Lett. 2006, 6, 2225–2231. (29) Shamsaie, A.; Heim, J.; Yanik, A. A.; Irudayaraj, J. Chem. Phys. Lett. 2008, 461, 131–135. (30) Kneipp, J.; Kneipp, H.; Rajadurai, A.; Redmond, R. W.; Kneipp, K. J. Raman Spectrosc. 2009, 40, 1–5. (31) Connor, E. E.; Mwamuka, J.; Gole, A.; Murphy, C. J.; Wyatt, M. D. Small 2005, 1, 325–327. (32) Tsoli, M.; Kuhn, H.; Brandau, W.; Esche, H.; Schmid, G. Small 2005, 1, 841–844. (33) Fujita, K.; Ishitobi, S.; Hamada, K.; Smith, N. I.; Taguchi, A.; Inouye, Y.; Kawata, S. J. Biomed. Opt. 2009, 14, 024038. (34) Shukla, R.; Bansal, V.; Chaudhary, M.; Basu, A.; Bhonde, R. R.; Sastry, M. Langmuir 2005, 21, 10644–10654. (35) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K. R.; Han, M. S.; Mirkin, C. A. Science 2006, 312, 1027–1030. (36) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. W. Nano Lett. 2006, 6, 662– 668. (37) Talley, C. E.; Jusinski, L.; Hollars, C. W.; Lane, S. M.; Huser, T. Anal. Chem. 2004, 76, 7064–7068.

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EXPERIMENTAL SECTION Cell Culture. CHO (Chinese hamster ovary) cells were obtained from ATCC/LGC Promochem (ATCC No. CCL-61, LGC Promochem, UK). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen Ltd.) without L-glutamine. The media was supplemented with fetal bovine serum (FBS, Invitrogen Ltd.) to a final concentration of 10% and L-glutamine to a final concentration of 4 mM. The cells were incubated in a humidified environment with 5% CO2 at 37 °C. At harvesting, old media was removed and cells were washed with PBS solution (8.1 mM Na2HPO4, 1.2 mM KH2PO4, 138 mM NaCl, 2.7 mM KCL, pH 7.4). Cells were detached from the culture flask with a trypsin/EDTA (ethylenediaminetetraacetic acid) solution (Invitrogen Ltd.). A stock solution of cells of ca. 4 × 106 cells per mL was obtained. Uptake of Nanoparticles into the Cells. Nanoparticles were synthesized using established methods38 and were delivered into live cells by passive uptake.34 In our work, typically, 0.5 mL of the stock solution of cells was added to a Nunclon T25 flask (with a surface area of 25 cm2) containing 5.0 mL of DMEM culture media and 100 µL of 63 nm (mean diameter) silver colloid. The mixture of cells, media, and colloidal was agitated for 10-15 min, prior to incubation at 37 °C, as described above, for 1 day. For labeling of the nanoparticles, typically 100 µL of 5 mM 4-mercaptobenzoic acid, 4-MBA, (in ethanol) was added into 1 mL of colloid solution (this amount of 4-MBA does cause some aggregation which is crucial to obtain good SERS signals) and allowed ca. 30 min to attach to the nanoparticles, prior to their introduction into the cell solution. SERS Measurements of Cells. For the SERS measurements, cells were washed exhaustively with PBS solutions, pH 7.4, before harvesting to ensure any nanoparticles loosely attached to the cell membrane were washed out. The harvested cells were centrifuged at 1000 rpm for 3 min to form a loose pellet of cells while the supernatant was subsequently decanted. The pellet of cells was resuspended in media, and the process was repeated several times. Finally, the cells were resuspended in PBS, pH 7.4. A small aliquot of cells was transferred to a thin quartz glass coverslip with an observation collar, to allow a sufficient reservoir of liquid to be contained. The cells were allowed to settle (adhere) for 60 min in an incubator at 37 °C. Once the cells were attached to the quartz surface, the reservoir was replenished with fresh PBS solution periodically. For the time-course experiments, cells were incubated with media containing nanoparticles in a Nunclon T25 flask and cells were sampled at intervals of 0, 2, 4, 8, 12, 16, 20, and 24 h. Ten cells were chosen randomly in each sample and were imaged at each time. For cell division experiments, cells were passaged and reincubated in a Nunclon T25 flask with nanoparticles over defined periods. They were allowed to grow for 24 h and were then harvested (each passage was defined as a new generation). Ten cells were chosen randomly and were imaged. Confocal Raman Microspectroscopy Measurements. All Raman spectra were acquired with a LabRam inverted microscope spectrometer, manufactured by Jobin Yvon Ltd. The spectrometer was equipped with multiple laser sources at wavelengths of 532 nm (70 mW), 633 nm (He-Ne laser, 20 mW), and 830 nm (diode (38) Leopold, N.; Lendl, B. J. Phys. Chem. B 2003, 107, 5723–5727.

Figure 1. High resolution SERS mapping of cells containing Ag nanoparticles. Figure 1A shows a brightfield image of a single CHO cell containing Ag nanoparticles with the SERS map overlaid (top). The SERS map alone is shown for clarity (bottom). Each pixel (green) corresponds to a 0.5 (x) × 0.5 (y) × 2.0 (z) micrometer confocal segment and represents the Raman spectrum from 700 to 1800 cm-1. Figure 1B shows examples of some of the individual SERS spectra (baseline corrected) extracted from the map shown in (A). Note that spectra differ as a consequence of the inhomogeneity of the cell.

laser) near IR laser, true confocal optics, a holographic transmission grating, and a charge coupled device (CCD) detector with 1024 × 256 pixels. The instrument included a precision motorized x-y-z sample stage for automated mapping at spatial resolution down to less than 1 µm in the x,y plane and ∼2 µm depth resolution and extensive software support (LabSpec 5) for data processing. In this study, 60× oil immersion (NA ) 1.25) and 100× objectives were used (U Plan FL, Nikon, Japan). This objective was mounted on a PI-721.10 piezo-actuator (Physik Instrumente, Germany) for automatic focusing of the microscope objective at different depths in the z-direction, enabling 3D mapping. A grating with 600 grooves mm-1, a confocal aperture of 300 µm, and an entrance slit of 150 µm were selected. The Raman spectrometer wavelength range was calibrated using the center frequency of the silicon band from a silicon sample (520.2 cm-1). Using these conditions, a typical acquisition time of 0.1-0.5 s was used with a laser power of 2 mW, to collect SERS spectra from cells. RESULTS AND DISCUSSION Under control conditions, with the short integration times described, no Raman signals were obtained from the CHO cells, in the absence of nanoparticles. Figure 1A shows a brightfield image of a single CHO cell containing Ag nanoparticles with the SERS map overlaid (top). The SERS map alone is shown for clarity (bottom). Each pixel (green) corresponds to a 0.5 (x) × 0.5 (y) × 2.0 (z) micrometer confocal segment. The image was constructed using the whole spectral region of 700-1800 cm-1 at 0.5 µm XY spacing over the selected area, typically incorporating the whole cell area and a background area. In this image, each pixel represents a single spectrum for the region. Typically, intracellular SERS signals are complex due to the nature of the surrounding environment. The intensities of signals are high, even at 0.5 s acquisition time.

Figure 2. SERS spectra of 4-MBA coated Ag nanoparticles in vitro (a) and (b); Both 4-MBA and cellular SERS signals from 4-MBA coated Ag nanoparticles inside CHO cells (c) and (d).

Figure 1B shows individual baseline corrected spectra obtained from the map shown in Figure 1A. Although each spectrum is different, due to the inhomogeneity of the cytosol, they share similar Raman peaks associated with proteins or amino acids (827, 998, 1133, 1358, 1474, 1625, and 1582 cm-1), lipids (1274, 1474, and 1625 cm-1), and DNA (827, 1214, and 1625 cm-1). Figure 2 shows the SERS spectra from the reporter molecule 4-MBA, attached to a nanoparticle, both inside and outside of the cells. 4-MBA has been used previously as an intracellular SERS label, to monitor pH.37,39 Consistent with the literature, we also obtained intense SERS signals from this reporter molecule. The SERS spectra recorded from 4-MBA outside the cells (in vitro) (39) Kneipp, J.; Kneipp, H.; Wittig, B.; Kneipp, K. J. Phys. Chem. C 2010, 114, 7421–7426.

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Figure 3. Time-course study of nanoparticle uptake by cells. CHO cells were incubated with 4-MBA coated nanoparticles for different lengths of time. At each time interval, 10 random cells were chosen and imaged using high resolution SERS spectroscopy. Figure 3A shows the SERS maps from a representative cell from each time interval (time in hours indicated by the number next to the SERS map shown). Each pixel (green) corresponds to a 0.5 (x) × 0.5 (y) × 2.0 (z) micrometer confocal segment at 1074 cm-1. The boundary of the cell in each case is represented by a broken white circle and is approximately 15 µm in diameter. Figure 3B illustrates the average SERS signal per unit area (as determined by SERS mapping) for each time interval.

shows two distinct spectra (Figure 2a,b): one with two prominent bands at 1074 and 1590 cm-1 (ring breathing mode of the benzene ring) and a weak band at 1184 cm-1; the other one shows three extra bands40 at 994, 1016, and 1568 cm-1. The intracellular SERS spectra contains characteristic cellular SERS information (with contributions from protein, lipids, and DNA similar to those described when bare Ag colloidal particles are used, Figure 1B), as well as a strong 4-MBA signal, which are clearly distinguishable in the spectra (including bands at 994, 1016, and 1568 cm-1, similar to the extracellular in vitro studies as shown in Figure 2a,b). Figure 3 shows the time-course study of SERS maps after incubation with Ag-4-MBA for 24 h. The Raman instrument provided a confocal optical slice whose depth in z was sufficient to provide a representative volume of the cell. All of these experiments were carried out in Nunclon T25 flasks to allow sufficient surface area for these adherent cells to behave normally. We also found that when attached to the Nunclon flask surface, it was easy to remove the nanoparticles which may have attached to the cell surface, by repeated washing. For the data presented, typically 10 cells were selected randomly for SERS mapping. To generate the SERS map using 4-MBA coated nanoparticles, the characteristic 1074 cm-1 band was selected to map the position of the nanoparticles (the other prominent 4-MBA band at 1590 cm-1 was occasionally obscured by bands originating from (40) Michota, A.; Bukowska, J. J. Raman Spectrosc. 2003, 34, 21–25.

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Figure 4. Monitoring the redistribution of intracellular nanoparticles after cell division via SERS spectroscopy. (A) Shows the average percentage of SERS activity (within the confocal volume), measured from 10 randomly chosen cells in each generation, following division. The inset to (A) shows the percentage of cells that did not show any SERS signal per cell generation. (B) Shows a typical SERS map from each generation (each pixel (green) corresponds to a 0.5 (x) × 0.5 (y) × 2.0 (z) micrometer confocal segment at 1074 cm-1). These results indicate that detectable SERS signals were present within the 4th generation of cells (i.e., after three rounds of cell division). No SERS signals could be detected beyond the fourth generation.

cellular components (see Figure 2c,d)). In Figure 3A, the boundary of the cell in each case is represented by a broken white circle and is approximately 15 µm in diameter (the overlaid SERS maps can be found in Supporting Information, Figure S1). After mixing the nanoparticles with the cells and exhaustive washing (0 h), cells do not show any SERS signals (showing that we can remove nanoparticles loosely attached to the cell membrane). After incubation with functionalized nanoparticles for 2 h, 90% of the cells showed SERS activity, although these were weak and poorly distributed signature maps (only 3% of the map gave an intracellular SERS signal). Cells imaged after between 2 and 16 h of incubation with Ag-4-MBA showed an increasing uptake of nanoparticles (after 4 h, all cells showed some SERS activity). After incubation for more than 16 h, the amount of nanoparticles taken into the cell did not further increase. Interestingly, although the cells have taken up large numbers of nanoparticles, it was observed that they continued to grow and divide. Figure 4A shows the percentage SERS signal within a confocal slice, recorded from 10 randomly mapped CHO cells per generation, for four generations of cells. Cells were incubated for 24 h with Ag-4-MBA and were washed. All cells (n ) 10, 100%) in generation 1 had a high percentage of SERS signal. The inset in Figure 4A shows that no cells had no nanoparticles within them. Figure 4B shows a typical SERS map from each generation (each pixel (green) corresponds to a 0.5 (x) × 0.5 (y) × 2.0 (z) micrometer confocal segment at 1074 cm-1). The boundary of the cell in each case is represented by a broken white circle and is approximately 15 µm in diameter (the overlaid SERS maps can be found in Supporting Information, Figure S2). The maps obtained after passage 1 (second generation) also show clear SERS signals, but the signal intensities and total signal coverage have fallen due to an increased distribution of the nanoparticles into the daughter cells, reducing the nanoparticle aggregation and hence signal. Cells (90%) still showed SERS signals (see inset Figure 1A). After passage 2 (third generation) and 3 (fourth generation), the intensity of SERS signals further decreased. For

Figure 5. Schematic representation of the nanoparticles uptake and subsequent redistribution by CHO cells. Note that in the uptake process the amount of nanoparticles increased and in cell division stages they decay dramatically.

example, at generation 4, the cell shows ca. 1% SERS signal per confocal slice (n ) 10). No SERS signals could be detected after the fourth passage. A Student’s t test was used to assess whether the distribution in consecutive generations was significantly different, given the error bars obtained. In all cases, p > 0.05. Similar cell division experiments were performed with bare Ag nanoparticles (see Supporting Information, Figure S3), indicating that the 4-MBA had no significant role other than to act as a clear reporter, making collection and, in particular, the mapping of the data more convenient. CONCLUSION We describe the applicability of surface-enhanced Raman scattering (SERS) spectroscopy to monitor silver nanoparticle uptake and their subsequent fate during cell culture over a number

of generations (see Figure 5 for a synopsis). While the toxicity of metal nanoparticles has been extensively studied, their fate over a number of generations has not been studied previously. Similarly, while some studies of intracellular nanoparticles using SERS spectroscopy have been reported, this is to our knowledge the first time that SERS spectroscopy has been employed to monitor the long-term behavior of intracellular nanoparticles in live cell populations. We found that the nanoparticles and the attached reporter molecules are stable over sufficiently long time periods to allow for quantitative detection and may pass to subsequent generations without significant loss of their SERS signals. The data presented in this work is a significant initial step in demonstrating the applicability of SERS spectroscopic detection for monitoring cellular behavior with intracellular nanoparticles over prolonged periods of time and illustrates the broader applicability of SERS detection in analytical chemistry and biological sciences.

ACKNOWLEDGMENT We would like to thank RCUK Basic Technology Grant (EPE032745\1) for funding this project (through the Molecular Nose project). Also, we thank Dr. Huabing Yin, Dr. Rab Wilson, and Dr. Norbert Klauke for useful discussions.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review June 4, 2010. Accepted July 26, 2010. AC101480T

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