Nano Research

, Volume 2, Issue 4, pp 279–291 | Cite as

In vivo therapeutic silencing of hypoxia-inducible factor 1 alpha (HIF-1α) using single-walled carbon nanotubes noncovalently coated with siRNA

  • Geoffrey Bartholomeusz
  • Paul Cherukuri
  • John Kingston
  • Laurent Cognet
  • Robert LemosJr.
  • Tonya K. Leeuw
  • Laura Gumbiner-Russo
  • R. Bruce Weisman
  • Garth Powis
Open Access
Research Article


A new approach is described for delivering small interfering RNA (siRNA) into cancer cells by noncovalently complexing unmodified siRNA with pristine single-walled carbon nanotubes (SWCNTs). The complexes were prepared by simple sonication of pristine SWCNTs in a solution of siRNA, which then served both as the cargo and as the suspending agent for the SWCNTs. When complexes containing siRNA targeted to hypoxia-inducible factor 1 alpha (HIF-1α) were added to cells growing in serum containing culture media, there was strong specific inhibition of cellular HIF-1 activity. The ability to obtain a biological response to SWCNT/siRNA complexes was seen in a wide variety of cancer cell types. Moreover, intratumoral administration of SWCNT- HIF-1α siRNA complexes in mice bearing MiaPaCa-2/HRE tumors significantly inhibited the activity of tumor HIF-1α. As elevated levels of HIF-1α are found in many human cancers and are associated with resistance to therapy and decreased patient survival, these results imply that SWCNT/siRNA complexes may have value as therapeutic agents.


siRNA single-walled carbon nanotubes anti-cancer therapy in vivo delivery agent 


  1. [1]
    Hoeckel, M.; Schlenger, K.; Hoeckel, S.; Vaupel, P. Hypoxic cervical cancers with low apoptotic index are highly aggressive. Cancer Res. 1999, 59, 4525–4528.Google Scholar
  2. [2]
    Brown, J. M. Exploiting the hypoxic cancer cell: Mechanisms and therapeutic strategies. Mol. Med. Today 2000, 6, 157–162.PubMedCrossRefGoogle Scholar
  3. [3]
    Brown, J. M. & Giaccia, A. J. The unique physiology of solid tumors: Opportunities (and problems) for cancer therapy. Cancer Res. 1998, 58, 1408–1416.PubMedGoogle Scholar
  4. [4]
    Unruh, A.; Ressel, A.; Mohamed, H. G.; Johnson, R. S.; Nadrowitz, R.; Richter, E.; Katschinski, D. M.; Wenger, R. H. The hypoxia-inducible factor-1α is a negative factor for tumor therapy. Oncogene 2003, 22, 3213–3220.PubMedCrossRefGoogle Scholar
  5. [5]
    Aebersold, D. M.; Burri, P.; Beer, K. T.; Laissue, J.; Djonov, V.; Greiner, R. H.; Semenza, G. L. Expression of hypoxia-inducible factor-1α: A novel predictive and prognostic parameter in the radiotherapy of oropharyngeal cancer. Cancer Res. 2001, 61, 2911–2916.PubMedGoogle Scholar
  6. [6]
    Zhang, X. W.; Kon, T.; Wang, H.; Li, F.; Huang, Q.; Rabbani, Z. N.; Kirkpatrick, J. P.; Vujaskovic, Z.; Dewhirst, M. W.; Li, C. Y. Enhancement of hypoxia-induced tumor cell death in vitro and radiation therapy in vivo by use of small interfering RNA targeted to hypoxia-inducible factor-1α. Cancer Res. 2004, 64, 8139–8142.PubMedCrossRefGoogle Scholar
  7. [7]
    Talks, K. L.; Turley, H.; Gatter, K. C.; Maxwell, P. H.; Pugh, C. W.; Ratcliffe, P. J.; Harris, A. L. The expression and distribution of the hypoxia-inducible factors HIF-1α and HIF-2α in normal human tissues, cancers, and tumor-associated macrophages. Am. J. Path. 2000, 157, 411–421.PubMedGoogle Scholar
  8. [8]
    Zhong, H.; De Marzo, A. M.; Laughner, E.; Lim, M.; Hilton, D. A.; Zagzag, D.; Buechler, P.; Isaacs, W. B.; Semenza, G. L.; Simons, J. W. Overexpression of hypoxia-inducible factor 1α in common human cancers and their metastases. Cancer Res. 1999, 59, 5830–5835.PubMedGoogle Scholar
  9. [9]
    Semenza, G. L. Targeting HIF-1α for cancer therapy. Nat. Rev. Cancer 2003, 3, 721–732.PubMedCrossRefGoogle Scholar
  10. [10]
    Liao, D.; Corle, C.; Seagroves, T. N.; Johnson, R. S. Hypoxia-inducible factor-1α is a key regulator of metastasis in a transgenic model of cancer initiation and progression. Cancer Res. 2007, 67, 563–572.PubMedCrossRefGoogle Scholar
  11. [11]
    Stoeltzing, O.; McCarty, M. F.; Wey, J. S.; Fan, F.; Liu, W. B.; Belcheva, A.; Bucana, C. D.; Semenza, G. L.; Ellis, L. M. Role of hypoxia-inducible factor 1α in gastric cancer cell growth, angiogenesis, and vessel maturation. J. Natl. Cancer Inst. 2004, 96, 946–956.PubMedCrossRefGoogle Scholar
  12. [12]
    Maxwell, P. H.; Dachs, G. U.; Gleadle, J. M.; Nicholls, L. G.; Harris, A. L.; Stratford, I. J.; Hankinson, O.; Pugh, C. W.; Ratcliffe, P. J. Hypoxia-inducible factor-1α modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc. Natl. Acad. Sci. USA 1997, 94, 8104–8109.PubMedCrossRefADSGoogle Scholar
  13. [13]
    Ryan, H. E.; Poloni, M.; McNulty, W.; Elson, D.; Gassmann, M.; Arbeit, J. M.; Johnson, R. S. Hypoxia-inducible factor-1α is a positive factor in solid tumor growth. Cancer Res. 2000, 60, 4010–4015.PubMedGoogle Scholar
  14. [14]
    Kung, A. L.; Wang, S.; Klco, J. M.; Kaelin, W. G.; Livingston, D. M. Suppresion of tumor growth through disruption of hypoxia-inducible transcription. Nat. Med. 2000, 6, 1335–1340.PubMedCrossRefGoogle Scholar
  15. [15]
    Dykxhoorn, D. M.; Lieberman, J. Knocking down diseases with siRNA. Cell 2006, 126, 231–235.PubMedCrossRefGoogle Scholar
  16. [16]
    Kam, N. W. S.; Dai, H. Carbon nanotubes as intracellular protein transporters: Generality and biological functionality. J. Am. Chem. Soc. 2005, 127, 6021–6026.PubMedCrossRefGoogle Scholar
  17. [17]
    Kam, N. W. S.; Liu, Z.; Dai, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J. Am. Chem. Soc. 2005, 127, 12492–12493.PubMedCrossRefGoogle Scholar
  18. [18]
    Kam, N. W. S.; Jessop, T. C.; Wender, P. A; Dai, H. Nanotube molecular transporters: Internalization of carbon nanotubes-protein conjugates into mammalian cells. J. Am. Chem. Soc. 2004, 126, 6850–6851.CrossRefGoogle Scholar
  19. [19]
    Zhang, Z. H.; Yang, X. Y.; Zhang, Y.; Zeng, B.; Wang, Z. J.; Zhu, T. H.; Roden, R. B. S.; Chen, Y. S.; Yang, R. C. Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth. Clin. Cancer Res. 2006, 12, 4933–4939.PubMedCrossRefGoogle Scholar
  20. [20]
    Cherukuri, P.; Bachilo, S. M.; Litovsky, S. H.; Weisman, R. B. Near-infrared flourescence microscopy of sinngled-walled carbon nanotubes in phagocytic cells. J. Am. Chem. Soc. 2004, 126, 15638–15639.PubMedCrossRefGoogle Scholar
  21. [21]
    Tasis, D.; Tagmatarchis, N.; Georgakilas, V.; Prato, M. Soluble carbon nanotubes. Chem. Eur. J. 2003, 9, 4000–4008.CrossRefGoogle Scholar
  22. [22]
    Bianco, A.; Kostarelos, K.; Prato, M. Application of carbon nanotubes in drug discovery. Curr. Opin. Chem. Biol. 2005, 9, 674–679.PubMedCrossRefGoogle Scholar
  23. [23]
    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
  24. [24]
    Tasis, D.; Tagmatarchis, N.; Bianco, A.; Prato, M. Chemistry of carbon nanotubes. Chem. Rev. 2006, 106, 1105–1136.PubMedCrossRefGoogle Scholar
  25. [25]
    Zhao, W.; Song, C.; Pehrsson, P. E. Water-soluble and optically pH-sensitive single-walled carbon nanotubes from surface modification. J. Am. Chem. Soc. 2002, 124, 12418–12419.PubMedCrossRefGoogle Scholar
  26. [26]
    He, P.; Urban, M. W. Controlled phospholipid functionalization of single-walled carbon nanotubes. Biomacromolecules 2005, 6, 2455–2457.PubMedCrossRefGoogle Scholar
  27. [27]
    Liu, Z.; Winters, M.; Holodniy, M.; Dai, H. siRNA delivery into human T cells and primary cells with carbonnanotube transporters. Angew. Chem. Int. Ed. 2007, 46, 1–6.CrossRefGoogle Scholar
  28. [28]
    Magrez, A.; Kasas, S.; Salicio, V.; Pasquier, N.; Seo, J. W.; Celio, M.; Catsicas, S.; Schwaller, B.; Forro, L. Cellular toxicity of carbon-based nanometrials. Nano Lett. 2006, 6, 1121–1125.PubMedCrossRefADSGoogle Scholar
  29. [29]
    Zheng, M.; Jagota, A.; Semke, E. D.; Diner, B. A.; Mclean, R. S.; Lustig, S. R.; Richardson, R. E.; Tassi, N. G. DNA-assisted dispersion and separation of carbon nanotubes. Nat. Mater. 2003, 2, 338–342.PubMedCrossRefADSGoogle Scholar
  30. [30]
    Dwyer, C.; Guthold, M.; Falvo, M.; Washburn, S.; Superfine, R.; Erie, D. DNA-functionalized single-walled carbon nanotubes. Nanotechnology 2002, 13, 601–604.CrossRefADSGoogle Scholar
  31. [31]
    Nepal, D.; Sohn, J. I.; Aicher, W. K.; Lee, S. J.; Geckeler, K. E. Supramolecular conjugates of carbon nanotubes and dna by a solid-state reaction. Biomacromolecules 2005, 6, 2919–2922.PubMedCrossRefGoogle Scholar
  32. [32]
    Zheng, M.; Jagota, A.; Strano, M. S.; Santos, A. P.; Barone, P.; Chou, S. G.; Diner, B. A.; Dresselhaus, M. S.; McLean, R. S.; Onoa, G. B.; Samsonidze, G. G.; Semke, E. D.; Usrey, M.; Walls, D. J. Structure-based carbon nanotubes sorting by sequence-dependent dna assembly. Science 2003, 302, 1545–1548.PubMedCrossRefADSGoogle Scholar
  33. [33]
    Nikolaev, P.; Bronikowski, M. J.; Bradley, R. K.; Rohmund, F.; Colbert, D. T.; Smith, K. A.; Smalley, R. E. Gas-phase catalytic growth of single-walled carbon nanotubes from carbon monoxide. Chem. Phys. Lett. 1999, 313, 91–97.CrossRefADSGoogle Scholar
  34. [34]
    Tsyboulski, D. A.; Bachilo, S. M.; Weisman, R. B. Versatile visualization of individual single-walled carbon nanotubes with near-infrared fluorescence microscopy. Nano Lett. 2005, 5, 975–979.PubMedCrossRefADSGoogle Scholar
  35. [35]
    PaineMurrieta, G. D.; Taylor, C. W.; Curtis, R. A.; Lopez, M. H. A.; Dorr, R. T.; Johnson, C. S.; Funk, C. Y.; Thompson, F.; Hersh, E. M. Human tumor models in the severe combined immune deficient (scid) mouse. Cancer Chemother Pharmacol. 1997, 40, 209–214.CrossRefGoogle Scholar
  36. [36]
    Svensson, R. U.; Barnes, J. M.; Rokhlin, O. W.; Cohen, M. B.; Henry, M. D. Chemotherapeutic agents up-regulate the cytomegalovirus promoter: implications for bioluminescence imaging of tumor response to therapy. Cancer Res. 2007, 67, 10445–10454.PubMedCrossRefGoogle Scholar
  37. [37]
    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. USA 2006, 103, 18882–18886.PubMedCrossRefADSGoogle Scholar
  38. [38]
    Singh, R.; Pantarotto, D.; Lacerda, L.; Pastorin, G.; Klumpp, C.; Prato, M.; Bianco, A.; Kostarelos, K. Tissue biodistribution and blood clearance rates of intravenously administered carbon nanotube radiotracers. Proc. Natl. Acad. Sci. USA 2006, 103, 3357–3362.PubMedCrossRefADSGoogle Scholar
  39. [39]
    Liu, Z.; Davis, C.; Cai, W. B.; He, L.; Chen, X. Y.; Dai, H. J. Circulation and long-term fate of functionalized, biocompatible single-walled carbon nanotubes in mice probed by Raman spectroscopy. Proc. Natl. Acad. Sci. USA 2008, 105, 1410–1415.PubMedCrossRefADSGoogle Scholar
  40. [40]
    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.PubMedCrossRefADSGoogle Scholar
  41. [41]
    Schipper, M. L.; Nakayama-Ratchford, N.; Davis, C. R.; Kam, N. W. S.; Chu, P.; Liu, Z.; Sun, X. M.; Dai, H. J.; Gambhir, S. S. A pilot toxicology study of single-walled carbon nanotubes in a small sample of mice. Nat. Nanotechnol. 2008, 3, 216–221.PubMedCrossRefGoogle Scholar
  42. [42]
    Yang, S. T.; Guo, W.; Lin, Y.; Deng, X. Y.; Wang, H. F.; Sun, H. F.; Liu, Y. F.; Wang, X.; Wang, W.; Chen, M.; Huang, Y. P.; Sun, Y. P. Biodistribution of pristine single-walled carbon nanotubes in vivo. J. Phys. Chem. 2007, 111, 17761–17764.Google Scholar
  43. [43]
    Thomas, C. E.; Ehrhardt, A.; Kay, M. A. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 2003, 4, 346–358.PubMedCrossRefGoogle Scholar
  44. [44]
    Fu, K.; Huang, W.; Lin, Y.; Zhang, D.; Hanks, T. W.; Rao, A. M.; Sun, Y. P. Functionalization of carbon nanotubes with bovine serum albumin in homogeneous aqueous soultions. J. Nanosci. Nanotech. 2002, 2, 457–461.CrossRefGoogle Scholar
  45. [45]
    Meng, J.; Song, L.; Xu, H.; Kong, H.; Wang, C.; Guo, X.; Xie, S. Effect of single-walled carbon nanotubes on the functions of plasma proteins and potentials in vascular prostheses. Nanomed. Nanotechnol. Biol. and Med. 2005, 1, 136–142.CrossRefGoogle Scholar

Copyright information

© Tsinghua University Press and Springer-Verlag GmbH 2009

Authors and Affiliations

  • Geoffrey Bartholomeusz
    • 1
  • Paul Cherukuri
    • 1
    • 2
  • John Kingston
    • 1
  • Laurent Cognet
    • 2
    • 3
  • Robert LemosJr.
    • 1
  • Tonya K. Leeuw
    • 2
  • Laura Gumbiner-Russo
    • 1
  • R. Bruce Weisman
    • 2
  • Garth Powis
    • 1
  1. 1.Department of Experimental TherapeuticsUniversity of Texas M. D. Anderson Cancer CenterHoustonUSA
  2. 2.Department of Chemistry, Center for Biological and Environmental Nanotechnology, and Institute of Biosciences and BioengineeringRice UniversityHoustonUSA
  3. 3.Centre de Physique Moléculaire Optique et HertzienneUniversité Bordeaux 1 and CNRSTalence CedexFrance

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