Nano Research

, Volume 5, Issue 4, pp 223–234 | Cite as

Cellular localization, accumulation and trafficking of double-walled carbon nanotubes in human prostate cancer cells

  • Vera Neves
  • Andreas Gerondopoulos
  • Elena Heister
  • Carmen Tîlmaciu
  • Emmanuel Flahaut
  • B. Soula
  • S. Ravi P. Silva
  • Johnjoe McFadden
  • Helen M. Coley
Research Article


Carbon nanotubes (CNTs) are at present being considered as potential nanovectors with the ability to deliver therapeutic cargoes into living cells. Previous studies established the ability of CNTs to enter cells and their therapeutic utility, but an appreciation of global intracellular trafficking associated with their cellular distribution has yet to be described. Despite the many aspects of the uptake mechanism of CNTs being studied, only a few studies have investigated internalization and fate of CNTs inside cells in detail. In the present study, intracellular localization and trafficking of RNA-wrapped, oxidized double-walled CNTs (oxDWNT-RNA) is presented. Fixed cells, previously exposed to oxDWNT-RNA, were subjected to immunocytochemical analysis using antibodies specific to proteins implicated in endocytosis; moreover cell compartment markers and pharmacological inhibitory conditions were also employed in this study. Our results revealed that an endocytic pathway is involved in the internalization of oxDWNT-RNA. The nanotubes were found in clathrin-coated vesicles, after which they appear to be sorted in early endosomes, followed by vesicular maturation, become located in lysosomes. Furthermore, we observed co-localization of oxDWNT-RNA with the small GTP-binding protein (Rab 11), involved in their recycling back to the plasma membrane via endosomes from the trans-golgi network.


Double-walled carbon nanotubes (DWNTs) intracellular localization uptake immunostaining inhibition of endocytosis 


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  1. [1]
    Pantarotto, D.; Briand, J. M.; Prato, M.; Bianco, A. Translocation of bioactive peptides across cell membranes by carbon nanotubes. Chem. Commun. 2004, 16–17.Google Scholar
  2. [2]
    Kostarelos, K.; Lacerda, L.; Pastorin, G.; Wu, W.; Wieckowski, S.; Luangsivilay, J.; Godefroy, S.; Pantarotto, D.; Briand, J. P.; Muller, S.; et al. Cellular uptake of functionalized carbon nanotubes is independent of functional group and cell type. Nat. Nanotechnol. 2007, 2, 108–113.CrossRefGoogle Scholar
  3. [3]
    Lacerda, L.; Raffa, S.; Prato, M.; Bianco, A.; Kostarelos, K. Cell-penetrating CNTs for delivery of therapeutics. Nano Today 2007, 2, 38–43.CrossRefGoogle Scholar
  4. [4]
    Neves, V.; Heister, E.; Costa, S.; Tîlmaciu, C.; Borowiak-Palen, E.; Giusca, C. E.; Flahaut, E.; Soula, B.; Coley, H. M.; McFadden, J.; et al. Uptake and release of double-walled carbon nanotubes by Mammalian cells. Adv. Funct. Mater. 2010, 20, 3272–3279.CrossRefGoogle Scholar
  5. [5]
    Kam, N. W. S.; Liu, Z.; Dai, H. J. Carbon nanotubes as intracellular transporters for proteins and DNA: An investigation of the uptake mechanism and pathway. Angew Chem. Int. Ed. 2006, 45, 577–581.CrossRefGoogle Scholar
  6. [6]
    Jin, H.; Heller, D. A.; Sharma, R.; Strano, M. S. Sizedependent cellular uptake and expulsion of single-walled carbon nanotubes: Single particle tracking and a generic uptake model for nanoparticles. ACS Nano 2009, 3, 149–158.CrossRefGoogle Scholar
  7. [7]
    Wei, M. L.; Bonzelius, R.; Scully, R. M.; Kelly, R. B.; Herman, G. A. GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells. J. Cell Biol. 1998, 140, 565–575.CrossRefGoogle Scholar
  8. [8]
    Connolly, C. N.; Futter, C. E,; Gibson, A.; Hopkins, C. R.; Cutler, D. F. Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase. J. Cell Biol. 1994, 127, 641–652.CrossRefGoogle Scholar
  9. [9]
    Yamashiro, D. J.; Tycko, B.; Fluss, S. R.; Maxfield, F. R. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell 1984, 37, 789–800.CrossRefGoogle Scholar
  10. [10]
    Mullock, B. M.; Bright, N. A.; Fearon, C. W.; Gray, S. R.; Luzio, J. Fusion of lysosomes with late endosomes produces a hybrid organelle of intermediate density and is NSF dependent. J. Cell Biol. 1998, 140, 591–601.CrossRefGoogle Scholar
  11. [11]
    Bucci, C.; Parton, R. G.; Mather, I. H.; Stunnenberg, H.; Simons, K.; Hoflack, B.; Zerial, M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell 1992, 70, 715–728.CrossRefGoogle Scholar
  12. [12]
    Ren, M.; Xu, G.; Zeng, J.; Lemos-Chiarandini, C. D.; Adesnik, M.; Sabatini, D. D. Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes. Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 6187–6192.CrossRefGoogle Scholar
  13. [13]
    Jin, H.; Heller D. A.; Strano, M. S. Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in NIH-3T3 cells. Nano Lett. 2008, 8, 1577–1585.CrossRefGoogle Scholar
  14. [14]
    Pantarotto, D.; Singh, R.; McCarthy, D.; Erhardt, M.; Briand, J. P.; Prato, M.; Kostarelos, K.; Bianco, A. Functionalized carbon nanotubes for plasmid DNA gene delivery. Angew Chem. Int. Ed. 2004, 43, 5242–5246.CrossRefGoogle Scholar
  15. [15]
    Mu, Q.; Broughton, D. L.; Yan, B. Endosomal leakage and nuclear translocation of multiwalled carbon nanotubes: developing a model for cell uptake. Nano Lett. 2009, 9, 4370–4375.CrossRefGoogle Scholar
  16. [16]
    Yehia, H. N.; Draper, R. K.; Mikoryak, C.; Walker, E. K.; Bajaj, P.; Musselman, I. H.; Daigrepont, M. C. Dieckmann, G. R.; Pantano, P. Single-walled carbon nanotube interactions with HeLa cells. J. Nanobiotechnol. 2007, 5, 3155–3163.CrossRefGoogle Scholar
  17. [17]
    Lacerda, L.; Pastorin, G.; Gathercole, D.; Buddle, J.; Prato, M.; Bianco, A.; Kostarelos, K. Intracellular trafficking of carbon nanotubes by confocal laser scanning microscopy. Adv. Mater. 2007, 19, 1780–1784.Google Scholar
  18. [18]
    Zhou, F.; Xing, D.; Wu, B.; Wu, S.; Ou, Z.; Chen, W. R. New insights of transmembranal mechanism and subcellular localization of noncovalently modified single-walled carbon nanotubes. Nano Lett. 2010, 10, 1677–1681.CrossRefGoogle Scholar
  19. [19]
    Saito, R.; Dresselhaus, G.; Dressehaus, M. S. Physical Properties of Carbon Nanotubes; London: Imperial College Press, 1998.CrossRefGoogle Scholar
  20. [20]
    Strano, M. S.; Doorn, S. K.; Haroz, E. H.; Kittrell, C.; Hauge, R. H.; Smalley, R. E. Assignment of (n, m) Raman and optical features of metallic single-walled carbon nanotubes. Nano Lett. 2003, 3, 1091–1096.CrossRefGoogle Scholar
  21. [21]
    Doorn, S. K.; Heller, D. A.; Barone, P. W.; Usrey, M. L.; Strano, M. S. Resonant Raman excitation profiles of individually dispersed single walled carbon nanotubes in solution. Appl. Phys. A 2004, 78, 1147–1155.CrossRefGoogle Scholar
  22. [22]
    Heister, E.; Lamprecht, C.; Neves, V.; Tîlmaciu, C.; Datas, L.; Flahaut, E.; Soula, B.; Hinterdorfer, P.; Coley, H. M.; Silva, S. R. P.; et al. Higher dispersion efficacy of functionalized carbon nanotubes in chemical and biological environments. ACS Nano 2010, 4, 2615–2626.CrossRefGoogle Scholar
  23. [23]
    Bartholomeusz, G.; Cherukuri, P.; Kingston, J.; Cognet, L.; Lemos, R.; Leeuw, T. K.; Gumbiner-Russo, L.; Weisman R. B.; Powis, G. In vivo therapeutic silencing of hypoxia-inducible factor 1 alpha (HIF-1alpha) using single-walled carbon nanotubes noncovalently coated with siRNA. Nano Res 2009, 2, 279–291.CrossRefGoogle Scholar
  24. [24]
    Wu, Y.; Phillips, J. A.; Liu, H.; Yang, R.; Tan, W. Carbon nanotubes protect DNA strands during cellular delivery. ACS Nano 2008, 2, 2023–2028.CrossRefGoogle Scholar
  25. [25]
    Aniento, F.; Emans, N.; Griffiths, G.; Gruenberg, J. Cytoplasmic dynein-dependent vesicular transport from early to late endosomes. J. Cell Biol. 1993, 123, 1373–1387.CrossRefGoogle Scholar
  26. [26]
    Parton, R. G.; Simons, K. The multiple faces of caveolae. Nat. Rev. Mol. Cell Biol. 2007, 8, 185–194.CrossRefGoogle Scholar
  27. [27]
    Luzio, J. P.; Mullock, B. M.; Pryor, P. R.; Lindsay, M. R.; James, D. E.; Piper, R. C. Relationship between endosomes and lysosomes. Biochem. Soc. Trans. 2001, 29, 476–480.CrossRefGoogle Scholar
  28. [28]
    Saftig, P.; Klumperman, J. Lysosome biogenesis and lysosomal membrane proteins: Trafficking meets function. Nat. Rev. Mol. Cell Biol. 2009, 10, 623–635.CrossRefGoogle Scholar
  29. [29]
    Ghosh, R. N.; Mallet, W. G.; Soe, T. T.; McGraw, T. E.; Maxfield, F. R. An endocytosed TGN38 chimeric protein is delivered to the TGN after trafficking through the endocytic recycling compartment in CHO cells. J. Cell Biol. 1998, 142, 923–936.CrossRefGoogle Scholar
  30. [30]
    Saraste, J.; Palade, G. E.; Farquhar, M. G. Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in exocrine pancreatic cells. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 6425–6429.CrossRefGoogle Scholar
  31. [31]
    Jones, A. T.; Clague, M. J. Phosphatidylinositol 3-kinase activity is required for early endosome fusion. Biochem. J. 1995, 311, 31–34.Google Scholar
  32. [32]
    Maxfield, F. R.; McGraw, T. E. Endocytic recycling. Nat. Rev. Mol. Cell Biol. 2004, 5, 121–132.CrossRefGoogle Scholar
  33. [33]
    Quon, M. J.; Chen, H.; Ing, B. L.; Liu, M. L.; Zarnowski, M. J.; Yonezawa, K.; Kasuga, M.; Cushman, S. W.; Taylor, S. I. Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells. Mol. Cell. Biol. 1995, 15, 5403–5411.Google Scholar
  34. [34]
    Clarke, J. F.; Young, P. W.; Yonezawa, K.; Kasuga, M.; Holman, G. D. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. Biochem. J. 1994, 300, 631–635.Google Scholar
  35. [35]
    Clague, M. J.; Thorpe, C.; Jones, A. T. Phosphatidylinositol 3-kinase regulation of fluid phase endocytosis. FEBS Lett. 1995, 367, 272–274.CrossRefGoogle Scholar
  36. [36]
    Li, G.; D’souza-Schorey, C.; Barbieri, M. A.; Roberts, R. L.; Klippel, A.; Williams, L. T.; Stahl P. D. Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 10207–10211.CrossRefGoogle Scholar
  37. [37]
    Brown, W. J.; DeWald, D. B.; Emr, S. D.; Plutner, H.; Balch, W. E. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J. Cell Biol. 1995, 130, 781–796.CrossRefGoogle Scholar
  38. [38]
    Davidson, H. W. Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes. J. Cell Biol. 1995, 130, 797–805.CrossRefGoogle Scholar
  39. [39]
    Cardone, M.; Mostov, K. Wortmannin inhibits transcytosis of dimeric IgA by the polymeric immunoglobulin receptor. FEBS Lett. 1995, 376, 74–76.CrossRefGoogle Scholar
  40. [40]
    Blommaart, E. F. C.; Krause, U.; Schellens, J. P. M.; Vreeling-Sindelarova, H.; Meijer, A. J. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem. 1997, 243, 240–246.CrossRefGoogle Scholar
  41. [41]
    Seglen, P. O.; Gordon, P. B. 3-Methyladenine: Specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes. Proc. Natl. Acad. Sci. U. S. A. 1982, 79, 1889–1892.CrossRefGoogle Scholar
  42. [42]
    Flahaut, E.; Bacsa, R.; Peigney, A.; Laurent, C. Gram-scale CCVD synthesis of double-walled carbon nanotubes. Chem. Commun. 2003, 1442–1443.Google Scholar
  43. [43]
    Heister, E.; Nevesa, V.; Tîlmaciub, C.; Lipertc, K.; Beltrána, V. S.; Coleya, H. M.; Silvad, S. R. P.; McFaddena, J. Triple functionalisation of single-walled carbon nanotubes with doxorubicin, a monoclonal antibody, and a fluorescent marker for targeted cancer therapy. Carbon 2009, 47, 2152–2160.CrossRefGoogle Scholar

Copyright information

© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Vera Neves
    • 1
    • 2
  • Andreas Gerondopoulos
    • 1
  • Elena Heister
    • 1
    • 2
  • Carmen Tîlmaciu
    • 3
  • Emmanuel Flahaut
    • 3
  • B. Soula
    • 3
  • S. Ravi P. Silva
    • 2
  • Johnjoe McFadden
    • 1
  • Helen M. Coley
    • 1
  1. 1.Faculty of Health and Medical SciencesUniversity of SurreyGuildfordUK
  2. 2.Nanoelectronics Centre, Advanced Technology InstituteUniversity of SurreyGuildfordUK
  3. 3.UPS/INP/CNRS, Institut Carnot CIRIMATUniversité de ToulouseToulouse, Cedex 9France

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