Abstract
Single-walled carbon nanotubes (SWNTs) with five different C13/C12 isotope compositions and well-separated Raman peaks have been synthesized and conjugated to five targeting ligands in order to impart molecular specificity. Multiplexed Raman imaging of live cells has been carried out by highly specific staining of cells with a five-color mixture of SWNTs. Ex vivo multiplexed Raman imaging of tumor samples uncovers a surprising up-regulation of epidermal growth factor receptor (EGFR) on LS174T colon cancer cells from cell culture to in vivo tumor growth. This is the first time five-color multiplexed molecular imaging has been performed in the near-infrared (NIR) region under a single laser excitation. Near zero interfering background of imaging is achieved due to the sharp Raman peaks unique to nanotubes over the low, smooth autofluorescence background of biological species.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Massoud, T. F.; Gambhir, S. S. Molecular imaging in living subjects: Seeing fundamental biological processes in a new light. Gene. Dev. 2003, 17, 545–580.
Wagnieres, G. A.; Star, W. M.; Wilson, B. C. In vivo fluorescence spectroscopy and imaging for oncological applications. Photochem. Photobiol. 1998, 68, 603–632.
Song, L. L.; Hennink, E. J.; Young, I. T.; Tanke, H. J. Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy. Biophys. J. 1995, 68, 2588–2600.
Aubin, J. E. Autofluorescence of viable cultured mammalian-cells. J. Histochem. Cytochem. 1979, 27, 36–43.
Frangioni, J. V. In vivo near-infrared fluorescence imaging. Curr. Opin. Chem. Biol. 2003, 7, 626–634.
Alivisatos, A. P.; Gu, W. W.; Larabell, C. Quantum dots as cellular probes. Ann. Rev. Biomed. Eng. 2005, 7, 55–76.
Xing, Y.; Chaudry, Q.; Shen, C.; Kong, K. Y.; Zhau, H. E.; Chung, L. W.; Petros, J. A.; O’Regan, R. M.; Yezhelyev, M. V.; Simons, J. W. et al. Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry. Nat. Protoc. 2007, 2, 1152–1165.
Fountaine, T. J.; Wincovitch, S. M.; Geho, D. H.; Garfield, S. H.; Pittaluga, S. Multispectral imaging of clinically relevant cellular targets in tonsil and lymphoid tissue using semiconductor quantum dots. Modern Pathol. 2006, 19, 1181–1191.
Nie, S. M.; Emery, S. R. Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 1997, 275, 1102–1106.
Cao, Y. W. C.; Jin, R. C.; Mirkin, C. A. Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 2002, 297, 1536–1540.
Keren, S.; Zavaleta, C.; Cheng, Z.; de la Zerda, A.; Gheysens, O.; Gambhir, S. S. Noninvasive molecular imaging of small living subjects using Raman spectroscopy. Proc. Nat. Acad. Sci. U. S. A. 2008, 105, 5844–5849.
Liu, Z.; Tabakman, S.; Welsher, K.; Dai, H. Carbon nanotubes in biology and medicine: In vitro and in vivo detection, imaging and drug delivery. Nano Res. 2009, 2, 85–120.
Liu, Z.; Cai, W.; He, L.; Nakayama, N.; Chen, K.; Sun, X.; Chen, X.; Dai, H. In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. Nat Nanotechnol. 2007, 2, 47–52.
Liu, Z.; Sun, X.; Nakayama, N.; Dai, H. Supramolecular chemistry on water-soluble carbon nanotubes for drug loading and delivery. ACS Nano 2007, 1, 50–56.
Feazell, R. P.; Nakayama-Ratchford, N.; Dai, H.; Lippard, S. J. Soluble single-walled carbon nanotubes as longboat delivery systems for platinum(IV) anticancer drug design. J. Am. Chem. Soc. 2007, 129, 8438–8439.
Moon, H. K.; Chang, C. I.; Lee, D. -K.; Choi, H. C. Effect of nucleases on the cellular internalization of fluorescent labeled DNA-functionalized single-walled carbon nanotubes. Nano Res. 2008, 1, 351–360.
Chen, R. J.; Bangsaruntip, S.; Drouvalakis, K. A.; Kam, N. W. S.; Shim, M.; Li, Y. M.; Kim, W.; Utz, P. J.; Dai, H. J. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors. Proc. Nat. Acad. Sci. U. S. A. 2003, 100, 4984–4989.
Chen, Z.; Tabakman, S. M.; Goodwin, A. P.; Kattah, M. G.; Daranciang, D.; Wang, X.; Zhang, G.; Li, X.; Liu, Z.; Utz, P. J. et al. Protein microarrays with carbon nanotubes as multicolor Raman labels. Nat. Biotechnol. 2008, 26, 1285–1292.
Welsher, K.; Liu, Z.; D, D.; Dai, H. Selective probing and imaging of cells with single walled carbon nanotubes as near-infrared fluorescent molecules. Nano Lett. 2008, 8, 586–590.
Cherukuri, P.; Gannon, C. J.; Leeuw, T. K.; Schmidt, H. K.; Smalley, R. E.; Curley, S. A.; Weisman, R. B. Mammalian pharmacokinetics of carbon nanotubes using intrinsic near-infrared fluorescence. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 18882–18886.
Hanlon, E. B.; Manoharan, R.; Koo, T. W.; Shafer, K. E.; Motz, J. T.; Fitzmaurice, M.; Kramer, J. R.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Prospects for in vivo Raman spectroscopy. Phys. Med. Biol. 2000, 45, R1–R59.
O’Connell, M. J.; Bachilo, S. M.; Huffman, C. B.; Moore, V. C.; Strano, M. S.; Haroz, E. H.; Rialon, K. L.; Boul, P. J.; Noon, W. H.; Kittrell, C. et al. Band gap fluorescence from individual single-walled carbon nanotubes. Science 2002, 297, 593–596.
Rao, A. M.; Richter, E.; Bandow, S.; Chase, B.; Eklund, P. C.; Williams, K. A.; Fang, S.; Subbaswamy, K. R.; Menon, M.; Thess, A. et al. Diameter-selective Raman scattering from vibrational modes in carbon nanotubes. Science 1997, 275, 187–191.
Liu, Z.; Li, X.; Tabakman, S. M.; Jiang, K.; Fan, S.; Dai, H. Multiplexed multi-color Raman imaging of live cells with isotopically modified single walled carbon nanotubes. J. Am. Chem. Soc. 2008, 130, 13540–13541.
Li, X. L.; Tu, X. M.; Zaric, S.; Welsher, K.; Seo, W. S.; Zhao, W.; Dai, H. J. Selective synthesis combined with chemical separation of single-walled carbon nanotubes for chirality selection. J. Am. Chem. Soc. 2007, 129, 15770–15771.
Liu, Z.; Davis, C.; Cai, W.; He, L.; Chen, X.; Dai, H. Circulation and long-term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by Raman spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 1410–1415.
Liu, Z.; Cai, W. B.; He, L. N.; Nakayama, N.; Chen, K.; Sun, X. M.; Chen, X. Y.; Dai, H. J. In vivo biodistribution and highly efficient tumour targeting of carbon nanotubes in mice. Nat. Nanotechnol. 2007, 2, 47–52.
Liu, Z.; Tabakman, S. M.; Chen, Z.; Dai, H. Preparation of carbon nanotube bioconjugates for biomedical applications. Nat. Protoc. 2009, 4, 1372–1382.
Lewis, G. D.; Figari, I.; Fendly, B.; Wong, W. L.; Carter, P.; Gorman, C.; Shepard, H. M. Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Cancer Immunol. Immun. 1993, 37, 255–263.
Haubner, R.; Wester, H. -J.; Weber, W. A.; Mang, C.; Ziegler, S. I.; Goodman, S. L.; Senekowitsch-Schmidtke, R.; Kessler, H.; Schwaiger, M. Noninvasive imaging of αvβ3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography. Cancer Res. 2001, 61, 1781–1785.
Cai, W.; Shin, D. -W.; Chen, K.; Gheysens, O.; Cao, Q.; Wang, S. X.; Gambhir, S. S.; Chen, X. Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects. Nano Lett. 2006, 6, 669–676.
Eliceiri, B. P.; Cheresh, D. A. The role of αv integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J. Clin. Invest. 1999, 103, 1227–1230.
Mulder, W. J. M.; Strijkers, G. J.; Habets, J. W.; Bleeker, E. J. W.; van der Schaft, D. W. J.; Storm, G.; Koning, G. A.; Griffioen, A. W.; Nicolay, K. MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle. FASEB J. 2005, 19, 2008–2010.
Bonner, J. A.; Buchsbaum, D. J.; Russo, S. M.; Fiveash, J. B.; Trummell, H. Q.; Curiel, D. T.; Raisch, K. P. Anti-EGFR-mediated radiosensitization as a result of augmented EGFR expression. Int. J. Radiat. Oncol. 2004, 59, 2–10.
Milenic, D. E.; Wong, K. J.; Baidoo, K. E.; Ray, G. L.; Garmestani, K.; Williams, M.; Brechbiel, M. W. Cetuximab: Preclinical evaluation of a monoclonal antibody targeting EGFR for radioimmunodiagnostic and radioimmunotherapeutic applications. Cancer Biother. Radio. 2008, 23, 619–631.
Milenic, D. E.; Garmestani, K.; Brady, E. D.; Albert, P. S.; Ma, D.; Abdulla, A.; Brechbiel, M. W. Targeting of HER2 antigen for the treatment of disseminated peritoneal disease. Clin. Cancer Res. 2004, 10, 7834–7841.
Oliveira, S.; Henegouwen, P. M. V. E.; Storm, G.; Schiffelers, R. M. Molecular biology of epidermal growth factor receptor inhibition for cancer therapy. Expert Opin. Biol. Th. 2006, 6, 605–617.
Heller, D. A.; Baik, S.; Eurell, T. E.; Strano, M. S. Single-walled carbon nanotube spectroscopy in live cells: Towards long-term labels and optical sensors. Adv. Mater. 2005, 17, 2793–2799.
Freudiger, C. W.; Min, W.; Saar, B. G.; Lu, S.; Holtom, G. R.; He, C.; Tsai, J. C.; Kang, J. X.; Xie, X. S. Label-free biomedical imaging with high sensitivity by stimulated Raman scattering microscopy. Science 2008, 322, 1857–1861.
Tu, X.; Zheng, M. A DNA-based approach to the carbon nanotube sorting problem. Nano Res. 2008, 1, 185–194.
Kam, N. W. S.; O’Connell, M.; Wisdom, J. A.; Dai, H. Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. Proc. Natl. Acad. Sci. USA 2005, 102, 11600–11605.
Author information
Authors and Affiliations
Corresponding author
Additional information
This article is published with open access at Springerlink.com
These authors contributed equally to the work.
Electronic supplementary material
Rights and permissions
Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License ( https://creativecommons.org/licenses/by-nc/2.0 ), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
About this article
Cite this article
Liu, Z., Tabakman, S., Sherlock, S. et al. Multiplexed five-color molecular imaging of cancer cells and tumor tissues with carbon nanotube Raman tags in the near-infrared. Nano Res. 3, 222–233 (2010). https://doi.org/10.1007/s12274-010-1025-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12274-010-1025-1