Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A novel intracellular protein delivery platform based on single-protein nanocapsules

Abstract

An average cell contains thousands of proteins that participate in normal cellular functions, and most diseases are somehow related to the malfunctioning of one or more of these proteins. Protein therapy1, which delivers proteins into the cell to replace the dysfunctional protein, is considered the most direct and safe approach for treating disease. However, the effectiveness of this method has been limited by its low delivery efficiency and poor stability against proteases in the cell, which digest the protein. Here, we show a novel delivery platform based on nanocapsules consisting of a protein core and a thin permeable polymeric shell that can be engineered to either degrade or remain stable at different pHs. Non-degradable capsules show long-term stability, whereas the degradable ones break down their shells, enabling the core protein to be active once inside the cells. Multiple proteins can be delivered to cells with high efficiency while maintaining low toxicity, suggesting potential applications in imaging, therapy and cosmetics fields.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Protein nanocapsules.
Figure 2: Transduction efficiency of protein nanocapsules in HeLa cells.
Figure 3: Stability and activity of nanocapsules in cells and mice.
Figure 4: Degradable nanocapsules.

References

  1. Birch, J. R. & Onakunle, Y. in Therapeutic Proteins, Methods and Protocols 1–16 (Humana Press, 2005).

    Book  Google Scholar 

  2. Wadia, J. S., Becker-Hapak, M. & Dowdy, S. F. Cell-Penetrating Peptides: Processes and Applications 365 (CRC Press, 2002).

    Google Scholar 

  3. Vyas, S. P., Singh, A. & Sihorkar, V. Ligand-receptor-mediated drug delivery: an emerging paradigm in cellular drug targeting. Crit. Rev. Ther. Drug Carrier Syst. 18, 1–76 (2001).

    Article  CAS  Google Scholar 

  4. Sato, H., Sugiyama, Y., Tsuji, A. & Horikoshi, I. Importance of receptor-mediated endocytosis in peptide delivery and targeting: kinetic aspects. Adv. Drug Deliv. Rev. 19, 445–467 (1996).

    Article  CAS  Google Scholar 

  5. Torchilin, V. P. Recent advances with liposomes as pharmaceutical carriers. Nature Rev. Drug Discov. 4, 145–160 (2005).

    Article  CAS  Google Scholar 

  6. Bulmus, V. et al. A new pH-responsive and glutathione-reactive, endosomal membrane-disruptive polymeric carrier for intracellular delivery of biomolecular drugs. J. Control Release 93, 105–120 (2003).

    Article  CAS  Google Scholar 

  7. Schwarze, S. R., Ho, A., Vocero-Akbani, A. & Dowdy, S. F. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285, 1569–1572 (1999).

    Article  CAS  Google Scholar 

  8. Heitz, F., Morris, M. C. & Divita, G. Twenty years of cell-penetrating peptides: from molecular mechanism to therapeutics. Br. J. Pharmacol. 157, 195–206 (2009).

    Article  CAS  Google Scholar 

  9. Gros, E. et al. A non-covalent peptide-based strategy for protein and peptide nucleic acid delivery. Biochim. Biophys. Acta 1758, 384–393 (2006).

    Article  CAS  Google Scholar 

  10. Fagain, C. O. Understanding and increasing protein stability. Biochim. Biophys. Acta 1252, 1–14 (1995).

    Article  Google Scholar 

  11. Hooper, N. M. Proteases in Biology and Medicine (Portland Press, 2002).

    Google Scholar 

  12. Brooks, H., Lebleu, B. & Vives, E. TAT peptide-mediated cellular delivery: back to basics. Adv. Drug Deliv. Rev. 57, 559–577 (2005).

    Article  CAS  Google Scholar 

  13. Poon, G. M. K. & Gariepy, J. Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells. Biochem. Soc. Trans. 35, 778–793 (2007).

    Google Scholar 

  14. Futami, J. et al. Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization. J. Biosci. Bioeng. 99, 95–103 (2005).

    Article  CAS  Google Scholar 

  15. Fischer, R., Kohler, K., Fotin-Mleczek, M. & Brock, R. A stepwise dissection of the intracellular fate of cationic cell-penetrating peptides. J. Biol. Chem. 279, 12625–12635 (2004).

    Article  CAS  Google Scholar 

  16. Akinc, A., Thomas, M., Klibanov, A. M. & Langer, R. Exploring polymethylenimine-mediated DNA transfection and the proton sponge hypothesis. J. Gene Med. 7, 657–663 (2004).

    Article  Google Scholar 

  17. Yamauchi, N. et al. Mechanism of synergistic cytotoxic effect between tumor-necrosis-factor and hyperthermia. Jpn J. Canc. Res. 83, 540–545 (1992).

    Article  CAS  Google Scholar 

  18. Kaliberov, S. A. et al. Combination of cytosine deaminase suicide gene expression with DR5 antibody treatment increases cancer cell cytotoxicity. Cancer Gene Ther. 13, 203–214 (2006).

    Article  CAS  Google Scholar 

  19. Folkes, L. K. & Wardman, P. Oxidative activation of indole-3-acetic acids to cytotoxic species — a potential new role for plant auxins in cancer therapy. Biochem. Pharmacol. 61, 129–136 (2001).

    Article  CAS  Google Scholar 

  20. de Melo, M. P., de Lima, T. M., Pithon-Curi, T. C. & Curi, R. The mechanism of indole acetic acid cytotoxicity. Toxicol. Lett. 148, 103–111 (2004).

    Article  Google Scholar 

  21. Fridovich, I. Superoxide radical: an endogenous toxicant. Ann. Rev. Pharmacol. Toxicol. 23, 239–257 (1983).

    Article  CAS  Google Scholar 

  22. Finkel, T. & Holbrook, N. J. Oxidants, oxidative stress and the biology of aging. Nature 408, 239–244 (2000).

    Article  CAS  Google Scholar 

  23. Ames, B. N., Shigenaga, M. K. & Hagen, T. M. Oxidants, antioxidants and the degenerative diseases of aging. Proc. Natl Acad. Sci. USA 90, 7915–7922 (1993).

    Article  CAS  Google Scholar 

  24. McCord, J. M. Superoxide dismutase in aging and disease: an overview. Methods Enzymol. 349, 331–341 (2002).

    Article  CAS  Google Scholar 

  25. Nicholson, D. W. et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376, 37–43 (1995).

    Article  CAS  Google Scholar 

  26. Porter, A. G. & Janicke, R. U. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6, 99–104 (1999).

    Article  CAS  Google Scholar 

  27. Oliver, L. & Vallette, F. M. The role of caspases in cell death and differentiation. Drug Resist. Updates 8, 163–170 (2005).

    Article  CAS  Google Scholar 

  28. Caron, N. J. et al. Intracellular delivery of a Tat–eGFP fusion protein into muscle cells. Mol. Ther. 3, 310–318 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was partially supported by the Defense Threat Reducing Agency (DTRA), NSF-CAREER, Sandia National Laboratories and CheungKong Scholar Program through Tsinghua University, China.

Author information

Authors and Affiliations

Authors

Contributions

M.Y., Z.L., T.S., Y.T. and Y.L. conceived and designed the experiments. M.Y., J.D. and Z.G. performed the experiments. Y.H., W.Z., L.W. and Z.H.Z. helped to analyse the data. Z.L., T.S., Y.T. and Y.L. co-wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Zheng Liu, Tatiana Segura, Yi Tang or Yunfeng Lu.

Supplementary information

Supplementary information

Supplementary information (PDF 1611 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yan, M., Du, J., Gu, Z. et al. A novel intracellular protein delivery platform based on single-protein nanocapsules. Nature Nanotech 5, 48–53 (2010). https://doi.org/10.1038/nnano.2009.341

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nnano.2009.341

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing