Enzyme-activatable polymer–drug conjugate augments tumour penetration and treatment efficacy

Abstract

A tumour microenvironment imposes barriers to the passive diffusion of molecules, which renders tumour penetration an unresolved obstacle to an effective anticancer drug delivery. Here, we present a γ-glutamyl transpeptidase-responsive camptothecin–polymer conjugate that actively infiltrates throughout the tumour tissue through transcytosis. When the conjugate passes on the luminal endothelial cells of the tumour blood vessels or extravasates into the tumour interstitium, the overexpressed γ-glutamyl transpeptidase on the cell membrane cleaves the γ-glutamyl moieties of the conjugate to generate positively charged primary amines. The resulting cationic conjugate undergoes caveolae-mediated endocytosis and transcytosis, which enables transendothelial and transcellular transport and a relatively uniform distribution throughout the tumour. The conjugate showed a potent antitumour activity in mouse models that led to the eradication of small solid tumours (~100 mm3) and regression of large established tumours with clinically relevant sizes (~500 mm3), and significantly extended the survival of orthotopic pancreatic tumour-bearing mice compared to that with the first-line chemotherapeutic drug gemcitabine.

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Fig. 1: Scheme and characterization of the enzyme-activatable polymer–drug nanomedicine.
Fig. 2: The cell cytotoxicity assays of polymer-drug conjugates.
Fig. 3: In vitro penetration of polymer–drug conjugates in HepG2 tumour spheroids.
Fig. 4: Blood clearance, biodistribution and tumour penetration of polymer–drug conjugates in HepG2-tumour-bearing mice.
Fig. 5: Antitumour efficacy of polymer–drug conjugates against subcutaneous HepG2 tumours.
Fig. 6: Antitumour activity of polymer–drug conjugates against orthotopic pancreatic tumours.

Data availability

The authors declare that all data generated or analysed during this study are available in this published article and its supplementary information files or from the corresponding author upon request.

References

  1. 1.

    Shi, J., Kantoff, P. W., Wooster, R. & Farokhzad, O. C. Cancer nanomedicine: progress, challenges and opportunities. Nat. Rev. Cancer 17, 20–37 (2017).

  2. 2.

    Youn, Y. S. & Bae, Y. H. Perspectives on the past, present, and future of cancer nanomedicine. Adv. Drug Deliv. Rev. 130, 3–11 (2018).

  3. 3.

    Sun, Q., Zhou, Z., Qiu, N. & Shen, Y. Rational design of cancer nanomedicine: nanoproperty integration and synchronization. Adv. Mater. 29, 1606628 (2017).

  4. 4.

    Matsumoto, Y. et al. Vascular bursts enhance permeability of tumour blood vessels and improve nanoparticle delivery. Nat. Nanotechnol. 11, 533–538 (2016).

  5. 5.

    Heldin, C. H., Rubin, K., Pietras, K. & Ostman, A. High interstitial fluid pressure—an obstacle in cancer therapy. Nat. Rev. Cancer 4, 806–813 (2004).

  6. 6.

    Flessner, M. F. et al. Resistance of tumor interstitial pressure to the penetration of intraperitoneally delivered antibodies into metastatic ovarian tumors. Clin. Cancer Res. 11, 3117–3125 (2005).

  7. 7.

    Minchinton, A. I. & Tannock, I. F. Drug penetration in solid tumours. Nat. Rev. Cancer 6, 583–592 (2006).

  8. 8.

    Trédan, O., Galmarini, C. M., Patel, K. & Tannock, I. F. Drug resistance and the solid tumor microenvironment. J. Natl Cancer Inst. 99, 1441–1454 (2007).

  9. 9.

    Blanco, E., Shen, H. & Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941–951 (2015).

  10. 10.

    Chauhan, V. P., Stylianopoulos, T., Boucher, Y. & Jain, R. K. Delivery of molecular and nanoscale medicine to tumors: transport barriers and strategies. Annu. Rev. Chem. Biomol. 2, 281–298 (2011).

  11. 11.

    Yuan, F. et al. Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft. Cancer Res. 54, 3352–3356 (1994).

  12. 12.

    Wong, C. et al. Multistage nanoparticle delivery system for deep penetration into tumor tissue. Proc. Natl Acad. Sci. USA 108, 2426–2431 (2011).

  13. 13.

    Oh, P. et al. Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung. Nat. Biotechnol. 25, 327–337 (2007).

  14. 14.

    Fung, K. Y. Y., Fairn, G. D. & Lee, W. L. Transcellular vesicular transport in epithelial and endothelial cells: challenges and opportunities. Traffic 19, 5–18 (2018).

  15. 15.

    Syvanen, S., Eden, D. & Sehlin, D. Cationization increases brain distribution of an amyloid-beta protofibril selective F(ab′)(2) fragment. Biochem. Biophys. Res. Commun. 493, 120–125 (2017).

  16. 16.

    Miura, S., Suzuki, H. & Bae, Y. H. A multilayered cell culture model for transport study in solid tumors: evaluation of tissue penetration of polyethyleneimine based cationic micelles. Nano Today 9, 695–704 (2014).

  17. 17.

    Feng, T. et al. Charge-convertible carbon dots for imaging-guided drug delivery with enhanced in vivo cancer therapeutic efficiency. ACS Nano 10, 4410–4420 (2016).

  18. 18.

    Xu, P. et al. Targeted charge-reversal nanoparticles for nuclear drug delivery. Angew. Chem. Int. Ed. 46, 4999–5002 (2007).

  19. 19.

    Gerweck, L. E. & Seetharaman, K. Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer. Cancer Res. 56, 1194–1198 (1996).

  20. 20.

    Li, H. J. et al. Smart superstructures with ultrahigh pH-sensitivity for targeting acidic tumor microenvironment: instantaneous size switching and improved tumor penetration. ACS Nano 10, 6753–6761 (2016).

  21. 21.

    Liu, Y. et al. Visualizing glioma margins by real-time tracking of γ-glutamyltranspeptidase activity. Biomaterials 173, 1–10 (2018).

  22. 22.

    Urano, Y. et al. Rapid cancer detection by topically spraying a γ-glutamyltranspeptidase-activated fluorescent probe. Sci. Transl. Med. 3, 110ra119 (2011).

  23. 23.

    Zhang, Q. et al. A new class of NO-donor pro-drugs triggered by γ-glutamyl transpeptidase with potential for reno-selective vasodilatation. Chem. Commun. 49, 1389–1391 (2013).

  24. 24.

    Castellano, I. & Merlino, A. γ-Glutamyltranspeptidases: sequence, structure, biochemical properties, and biotechnological applications. Cell. Mol. Life Sci. 69, 3381–3394 (2012).

  25. 25.

    Tate, S. S. & Meister, A. Interaction of γ-glutamyl transpeptidase with amino acids, dipeptides, and derivatives and analogs of glutathione. J. Biol. Chem. 249, 7593–7602 (1974).

  26. 26.

    Zhou, Z. et al. Charge-reversal drug conjugate for targeted cancer cell nuclear drug delivery. Adv. Funct. Mater. 19, 3580–3589 (2009).

  27. 27.

    Mason, J. E., Starke, R. D. & Van Kirk, J. E. Gamma-glutamyl transferase: a novel cardiovascular risk biomarker. Prev. Cardiol. 13, 36–41 (2010).

  28. 28.

    Yamada, J. et al. Elevated serum levels of alanine aminotransferase and gamma glutamyltransferase are markers of inflammation and oxidative stress independent of the metabolic syndrome. Atherosclerosis 189, 198–205 (2006).

  29. 29.

    Remaley, A. T. & Wilding, P. Macroenzymes: biochemical characterization, clinical significance, and laboratory detection. Clin. Chem. 35, 2261–2270 (1989).

  30. 30.

    Kwiecień, I. et al. The effect of modulation of γ-glutamyl transpeptidase and nitric oxide synthase activity on GSH homeostasis in HepG2 cells. Fundam. Clin. Pharmacol. 21, 95–103 (2007).

  31. 31.

    Hanigan, M. H. & Ricketts, W. A. Extracellular glutathione is a source of cysteine for cells that express gamma-glutamyl transpeptidase. Biochemistry 32, 6302–6306 (1993).

  32. 32.

    Han, L., Hiratake, J., Kamiyama, A. & Sakata, K. Design, synthesis, and evaluation of γ-phosphono diester analogues of glutamate as highly potent inhibitors and active site probes of γ-glutamyl transpeptidase. Biochemistry 46, 1432–1447 (2007).

  33. 33.

    Liao, Z.-X. et al. Mechanistic study of transfection of chitosan/DNA complexes coated by anionic poly(γ-glutamic acid). Biomaterials 33, 3306–3315 (2012).

  34. 34.

    Le, P. U. & Nabi, I. R. Distinct caveolae-mediated endocytic pathways target the Golgi apparatus and the endoplasmic reticulum. J. Cell Sci. 116, 1059–1071 (2003).

  35. 35.

    Wang, Z. et al. Delivery of nanoparticle-complexed drugs across the vascular endothelial barrier via caveolae. IUBMB Life 63, 659–667 (2011).

  36. 36.

    Bugno, J. et al. Size and surface charge of engineered poly(amidoamine) dendrimers modulate tumor accumulation and penetration: a model study using multicellular tumor spheroids. Mol. Pharm. 13, 2155–2163 (2016).

  37. 37.

    Suzuki, H. & Bae, Y. H. Evaluation of drug penetration with cationic micelles and their penetration mechanism using an in vitro tumor model. Biomaterials 98, 120–130 (2016).

  38. 38.

    Aller, S. G. et al. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 323, 1718–1722 (2009).

  39. 39.

    Palmeira, A., Sousa, E., Vasconcelos, M. H. & Pinto, M. M. Three decades of P-gp inhibitors: skimming through several generations and scaffolds. Curr. Med. Chem. 19, 1946–2025 (2012).

  40. 40.

    Lee, J. S. et al. Rhodamine efflux patterns predict P-glycoprotein substrates in the National Cancer Institute drug screens. Mol. Pharmacol. 46, 627–638 (1994).

  41. 41.

    Li, X.-Q. et al. Self-assembling nanomicelles of a novel camptothecin prodrug engineered with a redox-responsive release mechanism. Chem. Commun. 47, 8647–8649 (2011).

  42. 42.

    Shao, S. et al. A non-cytotoxic dendrimer with innate and potent anticancer and anti-metastatic activities. Nat. Biomed. Eng. 1, 745–757 (2017).

  43. 43.

    Wang, J. et al. The role of micelle size in tumor accumulation, penetration, and treatment. ACS Nano 9, 7195–7206 (2015).

  44. 44.

    Murakami, M. et al. Improving drug potency and efficacy by nanocarrier-mediated subcellular targeting. Sci. Transl. Med. 3, 64ra62 (2011).

  45. 45.

    Wushou, A. & Miao, X.-C. Tumor size predicts prognosis of head and neck synovial cell sarcoma. Oncol. Lett. 9, 381–386 (2015).

  46. 46.

    Hanigan, M. H., Frierson, H. F., Swanson, P. E. & De Young, B. R. Altered expression of gamma-glutamyl transpeptidase in human tumors. Hum. Pathol. 30, 300–305 (1999).

  47. 47.

    Lu, Y., Aimetti, A. A., Langer, R. & Gu, Z. Bioresponsive materials. Nat. Rev. Mater. 2, 16075 (2016).

  48. 48.

    Li, C.-L. et al. Survival advantages of multicellular spheroids vs. monolayers of HepG2 cells in vitro. Oncol. Rep. 20, 1465–1471 (2008).

  49. 49.

    Chai, M. G., Kim-Fuchs, C., Angst, E. & Sloan, E. K. Bioluminescent orthotopic model of pancreatic cancer progression. J. Vis. Exp. 76, e50395 (2013).

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Acknowledgements

We thank the National Natural Science Foundation of China (U1501243, 51833008, 51390481 and 51522304), National Basic Research Program of China (2014CB931900), the National Key Research and Development Program (2016YFA0200301), the Experimental Technology Research Program of Zhejiang University (SYB201605) and the Alfred P. Sloan Foundation for financial support.

Author information

Y.S. and Z.G. supervised the project and wrote the manuscript with Q.Z. and R.M.; Q.Z., C.X., S.S. and J.X. performed all the experiments; J.W., Q.Y., Z.Gan and R.M. re-evaluated the anticancer activity; Y.P. and X.L. instructed the bioassays; J.T. and Z.Z. instructed the synthesis.

Correspondence to Zhen Gu or Youqing Shen.

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Competing interests

Z.G. is a scientific co-founder of ZenCapsule, Inc. All the authors declare no conflicting interests.

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Supplementary Methods, Supplementary Figs. 1–30, Supplementary Table. 1 and 2, and Supplementary references.

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