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Enrichment-triggered prodrug activation demonstrated through mitochondria-targeted delivery of doxorubicin and carbon monoxide

Nature Chemistry volume 10, pages 787–794 (2018)Cite this article

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Abstract

Controlled activation is a critical component in prodrug development. Here we report a concentration-sensitive platform approach for bioorthogonal prodrug activation by taking advantage of reaction kinetics. Using two ‘click and release’ systems, we demonstrate enrichment and prodrug activation specifically in mitochondria to demonstrate the principle of the approach. In both cases, the payload (doxorubicin or carbon monoxide) was released inside the mitochondrial matrix following the enrichment-initiated click reaction. Furthermore, mitochondria-targeted delivery yielded substantial augmentation of functional biological and therapeutic effects in vitro and in vivo when compared to controls, which did not result in enrichment. This method is thus a platform for targeted drug delivery that is amenable to conjugation with a variety of molecules and is not limited to cell-surface delivery. Taken together, these two 'click and release' pairs clearly demonstrate the concept of enrichment-triggered drug release and the critical feasibility of treating clinically relevant diseases such as acute liver injury and cancer.

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Fig. 1: Schematic of the concept of enrichment-triggered prodrug activation.
Fig. 2: Cytotoxicity of click and release pairs in HeLa cells.
Fig. 3: Fluorescence imaging studies of the click reaction between 21 and 22 in RAW264.7 cells.
Fig. 4: CO prodrugs protect against acute liver failure.

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References

  1. Wang, B., Hu, L. & Siahaan, T. J. (eds) Drug Delivery: Principles and Applications 2nd edn (Drug Discovery and Development Series, Wiley, Hoboken 2016).

  2. He, X., Li, J., An, S. & Jiang, C. pH-sensitive drug-delivery systems for tumor targeting. Ther. Deliv. 4, 1499–1510 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Saravanakumar, G., Kim, J. & Kim, W. J. Reactive-oxygen-species-responsive drug delivery systems: promises and challenges. Adv. Sci. 4, 1600124 (2017).

    Article  CAS  Google Scholar 

  4. Quinn, J. F., Whittaker, M. R. & Davis, T. P. Glutathione responsive polymers and their application in drug delivery systems. Polym. Chem. 8, 97–126 (2017).

    Article  CAS  Google Scholar 

  5. Al-Nahain, A. et al. Triggered pH/redox responsive release of doxorubicin from prepared highly stable graphene with thiol grafted Pluronic. Int. J. Pharm. 450, 208–217 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Perez, C., Daniel, K. B. & Cohen, S. M. Evaluating prodrug strategies for esterase-triggered release of alcohols. ChemMedChem 8, 1662–1667 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zheng, Y. et al. An esterase-sensitive prodrug approach for controllable delivery of persulfide species. Angew. Chem. Int. Ed. 56, 11749–11753 (2017).

    Article  CAS  Google Scholar 

  8. Hamilton, J. R. & Trejo, J. Challenges and opportunities in protease-activated receptor drug development. Annu. Rev. Pharmacol. Toxicol. 57, 349–373 (2017).

    Article  CAS  PubMed  Google Scholar 

  9. Wiemer, A. J. & Wiemer, D. F. Prodrugs of phosphonates and phosphates: crossing the membrane barrier. Top. Curr. Chem. 360, 115–160 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Philip, A. K. & Philip, B. Colon targeted drug delivery systems: a review on primary and novel approaches. Oman Med. J. 25, 79–87 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Agarwal, P. & Bertozzi, C. R. Site-specific antibody–drug conjugates: the nexus of bioorthogonal chemistry, protein engineering, and drug development. Bioconjug. Chem. 26, 176–192 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Kern, J. C. et al. Discovery of pyrophosphate diesters as tunable, soluble, and bioorthogonal linkers for site-specific antibody–drug conjugates. J. Am. Chem. Soc. 138, 1430–1445 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Chudasama, V., Maruani, A. & Caddick, S. Recent advances in the construction of antibody–drug conjugates. Nat. Chem. 8, 114–119 (2016).

    Article  CAS  PubMed  Google Scholar 

  14. Zhao, X., Li, H. & Lee, R. J. Targeted drug delivery via folate receptors. Expert Opin. Drug Deliv. 5, 309–319 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Ahmed, M. & Narain, R. Carbohydrate-based materials for targeted delivery of drugs and genes to the liver. Nanomedicine (Lond.) 10, 2263–2288 (2015).

    Article  CAS  Google Scholar 

  16. Jin, W. et al. Discovery of PSMA-specific peptide ligands for targeted drug delivery. Int. J. Pharm. 513, 138–147 (2016).

    Article  CAS  PubMed  Google Scholar 

  17. Ji, X. et al. Click and release: a chemical strategy toward developing gasotransmitter prodrugs by using an intramolecular Diels–Alder reaction. Angew. Chem. Int. Ed. 55, 15846–15851 (2016).

    Article  CAS  Google Scholar 

  18. Wang, D. et al. A click-and-release approach to CO prodrugs. Chem. Commun. 50, 15890–15893 (2014).

    Article  CAS  Google Scholar 

  19. Rossin, R. et al. Triggered drug release from an antibody–drug conjugate using fast ‘click-to-release’ chemistry in mice. Bioconjug. Chem. 27, 1697–1706 (2016).

    Article  CAS  PubMed  Google Scholar 

  20. Versteegen, R. M. et al. Click to release: instantaneous doxorubicin elimination upon tetrazine ligation. Angew. Chem. Int. Ed. 52, 14112–14116 (2013).

    Article  CAS  Google Scholar 

  21. Matikonda, S. S. et al. Bioorthogonal prodrug activation driven by a strain-promoted 1,3-dipolar cycloaddition. Chem. Sci. 6, 1212–1218 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Ji, X. et al. Click and release: SO2 prodrugs with tunable release rates. Org. Lett. 19, 818–821 (2017).

    Article  CAS  PubMed  Google Scholar 

  23. Ji, X. et al. Click and fluoresce: a bioorthogonally activated smart probe for wash-free fluorescent labeling of biomolecules. J. Org. Chem. 82, 1471–1476 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Jimenez-Moreno, E. et al. Vinyl ether/tetrazine pair for the traceless release of alcohols in cells. Angew. Chem. Int. Ed. 56, 243–247 (2017).

    Article  CAS  Google Scholar 

  25. Steiger, A. K., Yang, Y., Royzen, M. & Pluth, M. D. Bio-orthogonal ‘click-and-release’ donation of caged carbonyl sulfide (COS) and hydrogen sulfide (H2S). Chem. Commun. 53, 1378–1380 (2017).

    Article  CAS  Google Scholar 

  26. Li, J., Jia, S. & Chen, P. R. Diels–Alder reaction-triggered bioorthogonal protein decaging in living cells. Nat. Chem. Biol. 10, 1003–1005 (2014).

    Article  CAS  PubMed  Google Scholar 

  27. Eising, S., Lelivelt, F. & Bonger, K. M. Vinylboronic acids as fast reacting, synthetically accessible, and stable bioorthogonal reactants in the Carboni–Lindsey reaction. Angew. Chem. Int. Ed. 55, 12243–12247 (2016).

    Article  CAS  Google Scholar 

  28. Luo, J., Liu, Q., Morihiro, K. & Deiters, A. Small-molecule control of protein function through Staudinger reduction. Nat. Chem. 8, 1027–1034 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Anseth, K. S. & Klok, H.-A. Click chemistry in biomaterials, nanomedicine, and drug delivery. Biomacromolecules 17, 1–3 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Kang, J., Hilmersson, G., Santamaría, J. & Rebek, J. Diels–Alder reactions through reversible encapsulation. J. Am. Chem. Soc. 120, 3650–3656 (1998).

    Article  CAS  Google Scholar 

  31. Murase, T., Horiuchi, S. & Fujita, M. Naphthalene Diels–Alder in a self-assembled molecular flask. J. Am. Chem. Soc. 132, 2866–2867 (2010).

    Article  CAS  PubMed  Google Scholar 

  32. Chen, W., Wang, D., Dai, C., Hamelberg, D. & Wang, B. Clicking 1,2,4,5-tetrazine and cyclooctynes with tunable reaction rates. Chem. Commun. 48, 1736–1738 (2012).

    Article  CAS  Google Scholar 

  33. Wang, D. et al. 3,6-Substituted-1,2,4,5-tetrazines: tuning reaction rates for staged labeling applications. Org. Biomol. Chem. 12, 3950–3955 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Shan, D., Nicolaou, M. G., Borchardt, R. T. & Wang, B. Prodrug strategies based on intramolecular cyclization reactions. J. Pharm. Sci. 86, 765–767 (1997).

    Article  CAS  PubMed  Google Scholar 

  35. Murphy, M. P. & Smith, R. A. J. Targeting antioxidants to mitochondria by conjugation to lipophilic cations. Ann. Rev. Pharmacol. Toxicol. 47, 629–656 (2007).

    Article  CAS  Google Scholar 

  36. Blackman, M. L., Royzen, M. & Fox, J. M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels–Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Devaraj, N. K., Weissleder, R. & Hilderbrand, S. A. Tetrazine-based cycloadditions: application to pretargeted live cell imaging. Bioconjug. Chem. 19, 2297–2299 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Smith, R. A. J., Porteous, C. M., Gane, A. M. & Murphy, M. P. Delivery of bioactive molecules to mitochondria in vivo. Proc. Natl Acad. Sci. USA 100, 5407–5412 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Janssen, M. J. H., Crommelin, D. J. A., Storm, G. & Hulshoff, A. Doxorubicin decomposition on storage. Effect of pH, type of buffer and liposome encapsulation. Int. J. Pharm. 23, 1–11 (1985).

    Article  CAS  Google Scholar 

  40. Otterbein, L. E. et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat. Med. 6, 422–428 (2000).

    Article  CAS  PubMed  Google Scholar 

  41. Otterbein, L. E. et al. Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nat. Med. 9, 183–190 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Klinger-Strobel, M. et al. Bactericidal effect of a photoresponsive carbon monoxide-releasing nonwoven against Staphylococcus aureus biofilms. Antimicrob. Agents Chemother. 60, 4037–4046 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Clark, J. E. et al. Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule. Circ. Res. 93, e2–e8 (2003).

    Article  CAS  PubMed  Google Scholar 

  44. Nagel, C. et al. Introducing [Mn(CO)3(tpa-κ3 N)]+ as a novel photoactivatable CO-releasing molecule with well-defined iCORM intermediates—synthesis, spectroscopy, and antibacterial activity. Dalton Trans. 43, 9986–9997 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Kuramitsu, K. et al. Carbon monoxide enhances early liver regeneration in mice after hepatectomy. Hepatology 53, 2016–2026 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Motterlini, R. & Foresti, R. Biological signaling by carbon monoxide and carbon monoxide-releasing molecules. Am. J. Physiol. Cell. Physiol. 312, C302–C313 (2017).

    Article  PubMed  Google Scholar 

  47. Motterlini, R. et al. Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities. Circ. Res. 90, E17–E24 (2002).

    Article  CAS  PubMed  Google Scholar 

  48. Pan, Z. et al. Organic CO prodrugs: structure–CO-release rate relationship studies. Chem. Eur. J. 23, 9838–9845 (2017).

    Article  CAS  PubMed  Google Scholar 

  49. Hernandez-Aguilera, A. et al. Mitochondrial dysfunction: a basic mechanism in inflammation-related non-communicable diseases and therapeutic opportunities. Mediat. Inflamm. 2013, 135698 (2013).

    Article  CAS  Google Scholar 

  50. Sawle, P. et al. Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages. Br. J. Pharmacol. 145, 800–810 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Schallner, N. & Otterbein, L. E. Friend or foe? Carbon monoxide and the mitochondria. Front. Physiol. 6, 17 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Marques, A. R. et al. Generation of carbon monoxide releasing molecules (CO-RMs) as drug candidates for the treatment of acute liver injury: targeting of CO-RMs to the liver. Organometallics 31, 5810–5822 (2012).

    Article  CAS  Google Scholar 

  53. Zuckerbraun, B. S. et al. Carbon monoxide protects against liver failure through nitric oxide-induced heme oxygenase 1. J. Exp. Med. 198, 1707–1716 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Motterlini, R. & Otterbein, L. E. The therapeutic potential of carbon monoxide. Nat. Rev. Drug Discov. 9, 728–743 (2010).

    Article  CAS  PubMed  Google Scholar 

  55. Robinson, M. W., Harmon, C. & O’Farrelly, C. Liver immunology and its role in inflammation and homeostasis. Cell. Mol. Immunol. 13, 267–276 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Bradley, M. J. et al. Host responses to concurrent combined injuries in non-human primates. J. Inflamm. 14, 23 (2017).

    Article  Google Scholar 

  57. Queiroga, C. S. F., Almeida, A. S. & Vieira, H. L. A. Carbon monoxide targeting mitochondria. Biochem. Res. Int. 2012, 749845 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors thank Z. Liu at the Center for Molecular and Translational Medicine at Georgia State University for taking confocal images. The authors also acknowledge GSU Molecular Basis of Disease (Z.P. and M.R.C.), Brains & Behaviors (B.Y.) and University 2CI (L.K.C.d.l.C.) fellowships for their support, and the Department of Defense Peer Reviewed Medical Research Program (W81XWH-16-0464 and NIH R44 DK111260-02 to L.E.O.).

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Author notes

  1. These authors contributed equally: Yueqin Zheng, Xingyue Ji.

Authors and Affiliations

  1. Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA

    Yueqin Zheng, Xingyue Ji, Bingchen Yu, Kaili Ji, Mengyuan Zhu, Manjusha Roy Choudhury, Ladie Kimberly C. De La Cruz, Vayou Chittavong, Zhixiang Pan, Zhengnan Yuan & Binghe Wang

  2. Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA, USA

    Yueqin Zheng, Xingyue Ji, Bingchen Yu, Kaili Ji, Mengyuan Zhu, Manjusha Roy Choudhury, Ladie Kimberly C. De La Cruz, Vayou Chittavong, Zhixiang Pan, Zhengnan Yuan & Binghe Wang

  3. Harvard Medical School, Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA, USA

    David Gallo, Eva Csizmadia & Leo E. Otterbein

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  1. Yueqin Zheng
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Contributions

B.W. conceived the initial idea, and supervised the study and the overall manuscript preparation and revision process. B.W. also revised the manuscript. Y.Z. and X.J. designed and performed the experiments, and drafted the manuscript. B.Y. and K.J. conducted the in vitro biology studies. D.G. and E.C. performed the in vivo studies. M.Z carried out the computational work. M.R.C., L.K.C.d.l.D., V.C., Z.Y., and Z.P. conducted studies of the reaction kinetics and some of the synthesis. L.E.O supervised and/or performed the in vivo studies, analysed and interpreted the data, and revised the manuscript. All authors reviewed, edited and approved the manuscript.

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Correspondence to Leo E. Otterbein or Binghe Wang.

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L.E.O. is a scientific consultant for Hillhurst Biopharmaceuticals. All other authors declare no competing interests.

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Zheng, Y., Ji, X., Yu, B. et al. Enrichment-triggered prodrug activation demonstrated through mitochondria-targeted delivery of doxorubicin and carbon monoxide. Nature Chem 10, 787–794 (2018). https://doi.org/10.1038/s41557-018-0055-2

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