Advertisement

Pharmaceutical Research

, Volume 32, Issue 4, pp 1354–1367 | Cite as

Short-Chain Glycoceramides Promote Intracellular Mitoxantrone Delivery from Novel Nanoliposomes into Breast Cancer Cells

  • Lília R. Cordeiro Pedrosa
  • Timo L. M. ten Hagen
  • Regine Süss
  • Albert van Hell
  • Alexander M. M. Eggermont
  • Marcel Verheij
  • Gerben A. Koning
Research Paper

Abstract

Purpose

To improve therapeutic activity of mitoxantrone (MTO)-based chemotherapy by reducing toxicity through encapsulation in nanoliposomes and enhancing intracellular drug delivery using short-chain sphingolipid (SCS) mediated tumor cell membrane permeabilization.

Methods

Standard (MTOL) and nanoliposomes enriched with the SCS, C8-Glucosylceramide or C8-Galactosylceramide (SCS-MTOL) were loaded by a transmembrane ammonium sulphate gradient and characterized by DLS and cryo-TEM. Intracellular MTO delivery was measured by flow cytometry and imaged by fluorescence microscopy. In vitro cytotoxicity was studied in breast carcinoma cell lines. Additionally, live cell confocal microscopy addressed the drug delivery mechanism by following the intracellular fate of the nanoliposomes, the SCS and MTO. Intratumoral MTO localization in relation to CD31-positive tumor vessels and CD11b positive cells was studied in an orthotopic MCF-7 breast cancer xenograft.

Results

Stable SCS-MTOL were developed increasing MTO delivery and cytotoxicity to tumor cells compared to standard MTOL. This effect was much less pronounced in normal cells. The drug delivery mechanism involved a transfer of SCS to the cell membrane, independently of drug transfer and not involving nanoliposome internalization. MTO was detected intratumorally upon MTOL and SCS-MTOL treatment, but not after free MTO, suggesting an important improvement in tumor drug delivery by nanoliposomal formulation. Nanoliposomal MTO delivery and cellular uptake was heterogeneous throughout the tumor and clearly correlated with CD31-positive tumor vessels. Yet, MTO uptake by CD11b positive cells in tumor stroma was minor.

Conclusions

Nanoliposomal encapsulation improves intratumoral MTO delivery over free drug. Liposome bilayer-incorporated SCS preferentially permeabilize tumor cell membranes enhancing intracellular MTO delivery.

KEY WORDS

Mitoxantrone Chemotherapy Short-chain sphingolipids Tumor-cell membrane-permeability modulation Targeting tumor cell membrane 

Notes

ACKNOWLEDGEMENTS AND DISCLOSURES

This work was financed by the Dutch Cancer Society. The authors thank Thomas Soullié for technical assistance with various aspects of histology and data processing and Sabine Barnert for performing cryo-TEM analyses.

Supplementary material

11095_2014_1539_MOESM1_ESM.doc (32 kb)
Supplemental Table 1 (DOC 32 kb)

References

  1. 1.
    Dunn CJ, Goa KL. Mitoxantrone: a review of its pharmacological properties and use in acute nonlymphoblastic leukaemia. Drugs Aging. 1996;9(2):122–47.CrossRefPubMedGoogle Scholar
  2. 2.
    White RJ, Durr FE. Development of mitoxantrone. Investig New Drugs. 1985;3(2):85–93.CrossRefGoogle Scholar
  3. 3.
    Koutinos G, Stathopoulos GP, Dontas I, et al. The effect of doxorubicin and its analogue mitoxantrone on cardiac muscle and on serum lipids: an experimental study. Anticancer Res. 2002;22(2A):815–20.PubMedGoogle Scholar
  4. 4.
    Chungun A, Uchide T, Tsurimaki C, et al. Mechanisms responsible for reduced cardiotoxicity of mitoxantrone compared to doxorubicin examined in isolated guinea-pig heart preparations. J Vet Med Sci. 2008;70:255–64.CrossRefGoogle Scholar
  5. 5.
    Murray TJ. The cardiac effects of mitoxantrone: do the benefits in multiple sclerosis outweigh the risks? Expert Opin Drug Saf. 2006;5(2):265–74.CrossRefPubMedGoogle Scholar
  6. 6.
    Cristofanilli M, Holmes F, Esparza L, et al. Phase I/II trial of high dose mitoxantrone in metastatic breast cancer: the M.D. Anderson Cancer Center experience. Breast Cancer Res Treat. 1999;54(3):225–33.CrossRefPubMedGoogle Scholar
  7. 7.
    Pusztai L, Holmes FA, Fraschini G, Hortobagyi GN. Phase II study of mitoxantrone by 14-day continuous infusion with granulocyte colony-stimulating factor (GCSF) support in patients with metastatic breast cancer and limited prior therapy. Cancer Chemother Pharmacol. 1999;43(1):86–91.CrossRefPubMedGoogle Scholar
  8. 8.
    Cook AM, Chambers EJ, Rees GJG. Comparison of mitozantrone and epirubicin in advanced breast cancer. Clin Oncol. 1996;8:363–6.CrossRefGoogle Scholar
  9. 9.
    Faulds D, Balfour J, Chrisp C, Langtry D. Mitoxantrone. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in the chemotherapy of cancer. Drugs. 1991;41(3):400–49.CrossRefPubMedGoogle Scholar
  10. 10.
    Fox EJ. Mechanism of action of mitoxantrone. Neurology. 2004;63(12 suppl 6):S15–8.CrossRefPubMedGoogle Scholar
  11. 11.
    Feofanov A, Sharonov S, Kudelina I, Fleury F, Nabiev I. Localization and molecular interactions of mitoxantrone within living K562 cells as probed by confocal spectral imaging analysis. Biophys J. 1997;73(6):3317–27.CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Hajihassan Z, Rabbani-Chadegani A. Studies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteins. J Biomed Sci. 2009;16(31).Google Scholar
  13. 13.
    van Dalen EC, van der Pal HJH, Bakker PJM, Caron HN, Kremer LCM. Cumulative incidence and risk factors of mitoxantrone-induced cardiotoxicity in children: a systematic review. Eur J Cancer. 2004;40(5):643–52.CrossRefPubMedGoogle Scholar
  14. 14.
    Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev. 2013;65(1):36–48.CrossRefPubMedGoogle Scholar
  15. 15.
    Paliwal SR, Paliwal R, Agrawal GP, Vyas SP. Liposomal nanomedicine for breast cancer therapy. Nanomedicine (London). 2011;6(6):1085–100.CrossRefGoogle Scholar
  16. 16.
    Koning GA, Krijger GC. Targeted multifunctional lipid-based nanocarriers for image-guided drug delivery. Anti Cancer Agents Med Chem. 2007;7(4):425–40.CrossRefGoogle Scholar
  17. 17.
    Mattheolabakis G, Rigas B, Constantinides PP. Nanodelivery strategies in cancer chemotherapy: biological rationale and pharmaceutical perspectives. Nanomedicine (London). 2012;7(10):1577–90.CrossRefGoogle Scholar
  18. 18.
    Deshpande PP, Biswas S, Torchilin VP. Current trends in the use of liposomes for tumor targeting. Nanomedicine (London). 2013;8(9):1509–28.CrossRefGoogle Scholar
  19. 19.
    Seynhaeve AL, Dicheva BM, Hoving S, Koning GA, Ten Hagen TL. Intact doxil is taken up intracellularly and released doxorubicin sequesters in the lysosome: evaluated by in vitro/in vivo live cell imaging. J Control Release. 2013;172(1):330–40.CrossRefPubMedGoogle Scholar
  20. 20.
    Seynhaeve ALB, Hoving S, Schipper D, et al. Tumor necrosis factor α mediates homogeneous distribution of liposomes in murine melanoma that contributes to a better tumor response. Cancer Res. 2007;67(19):9455–62.CrossRefPubMedGoogle Scholar
  21. 21.
    O’Brien MER, Wigler N, Inbar M, et al. Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYXâ“¢/Doxil®) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann Oncol. 2004;15(3):440–9.CrossRefPubMedGoogle Scholar
  22. 22.
    Laginha KM, Verwoert S, Charrois GJR, Allen TM. Determination of doxorubicin levels in whole tumor and tumor nuclei in murine breast cancer tumors. Clin Cancer Res. 2005;11(19 Pt1):6944–9.CrossRefPubMedGoogle Scholar
  23. 23.
    Visani G, Isidori A. Doxorubicin variants for hematological malignancies. Nanomedicine (London). 2011;6(2):303–6.CrossRefGoogle Scholar
  24. 24.
    van Lummel M, van Blitterswijk WJ, Vink SR, 1, et al. Enriching lipid nanovesicles with short-chain glucosylceramide improves doxorubicin delivery and efficacy in solid tumors. FASEB J. 2009;25:280–9.CrossRefGoogle Scholar
  25. 25.
    Veldman RJ, Zerp S, van Blitterswijk WJ, Verheij M. N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx. Br J Cancer. 2004;90(4):917–25.CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Veldman RJ, Zerp S, van Blitterswijk WJ, et al. Coformulated N-octanoyl-glucosylceramide improves cellular delivery and cytotoxicity of liposomal doxorubicin. J Pharmacol Exp Ther. 2005;315(2):704–10.CrossRefPubMedGoogle Scholar
  27. 27.
    Pedrosa LRC, Hell A, Suss R, et al. Improving intracellular doxorubicin delivery through nanoliposomes equipped with selective tumor cell membrane permeabilizing short-chain sphingolipids. Pharm Res. 2013;30(7):1883–95.CrossRefPubMedGoogle Scholar
  28. 28.
    van Hell AJ, Melo MN, van Blitterswijk WJ, et al. Defined lipid analogues induce transient channels to facilitate drug-membrane traversal and circumvent cancer therapy resistance. Sci Rep. 2013;3:1949.PubMedCentralPubMedGoogle Scholar
  29. 29.
    Siskind LJ, Fluss S, Bui M, Colombini M. Sphingosine forms channels in membranes that differ greatly from those formed by ceramide. J Bioenerg Biomembr. 2005;37(4):227–36.CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Haran G, Cohen R, Bar LK, Barenholz Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim Biophys Acta. 1993;1151(2):201–15.CrossRefPubMedGoogle Scholar
  31. 31.
    Pinto AC, Moreira JN, Simões S. Liposomal imatinib-mitoxantrone combination: formulation development and therapeutic evaluation in an animal model of prostate cancer. Prostate. 2011;71(1):81–90.CrossRefPubMedGoogle Scholar
  32. 32.
    Li C, Cui J, Wang C, et al. Encapsulation of mitoxantrone into pegylated SUVs enhances its antineoplastic efficacy. Eur J Pharm Biopharm. 2008;70(2):657–65.CrossRefPubMedGoogle Scholar
  33. 33.
    Lim HJ, Masin D, Madden TD, Bally MB. Influence of drug release characteristics on the therapeutic activity of liposomal mitoxantrone. J Pharmacol Exp Ther. 1997;281(1):566–73.PubMedGoogle Scholar
  34. 34.
    Rouser G, Fkeischer S, Yamamoto A. Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970;5(5):494–6.CrossRefPubMedGoogle Scholar
  35. 35.
    Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973;52(11):2745–56.CrossRefPubMedCentralPubMedGoogle Scholar
  36. 36.
    Skehan P, Storeng R, Scudiero D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990;82(13):1107–12.CrossRefPubMedGoogle Scholar
  37. 37.
    Li L, ten Hagen TLM, Schipper D, 2, et al. Triggered content release from optimized stealth thermosensitive liposomes using mild hyperthermia. J Control Release. 2010;143:274–9.CrossRefPubMedGoogle Scholar
  38. 38.
    Li X, Hirsh DJ, Cabral-Lilly D, 1, et al. Doxorubicin physical state in solution and inside liposomes loaded via a pH gradient. Biochim Biophys Acta. 1998;1415:23–40.CrossRefPubMedGoogle Scholar
  39. 39.
    Law SL, Chang P, Lin CH. Characteristics of mitoxantrone loading on liposomes. Int J Pharm. 1991;70:1–7.CrossRefGoogle Scholar
  40. 40.
    Durr FE, Wallace RE, Citarella RV. Molecular and biochemical pharmacology of mitoxantrone. Cancer Treat Rev. 1983;10(Suppl B):3–11.CrossRefPubMedGoogle Scholar
  41. 41.
    Orthmann A, Zeisig R, Suss R, et al. Treatment of experimental brain metastasis with MTO-liposomes: impact of fluidity and LRP-targeting on the therapeutic result. Pharm Res. 2012;29(7):1949–59.CrossRefPubMedGoogle Scholar
  42. 42.
    Maeda H, Nakamura H, Fang J. The EPR effect for macromolecular drug delivery to solid tumors: improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv Drug Deliv Rev. 2013;65(1):71–9.CrossRefPubMedGoogle Scholar
  43. 43.
    Yuan F, Leunig M, Huang SK, Berk DA, Papahadjopoulos D, Jain RK. Microvascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft. Cancer Res. 1994;54(13):3352–6.PubMedGoogle Scholar
  44. 44.
    Nakasone ES, Askautrud HA, Kees T, et al. Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance. Cancer Cell. 2012;21(4):488–503.CrossRefPubMedCentralPubMedGoogle Scholar
  45. 45.
    Li L, ten Hagen TLM, Schipper D, et al. Improved intratumoral nanoparticle extravasation and penetration by mild hyperthermia. J Control Release. 2013;167(2):130–7.CrossRefPubMedGoogle Scholar
  46. 46.
    Storm G, Steerenberg PA, Emmen F, van Borssum WM, Crommelin DJ. Release of doxorubicin from peritoneal macrophages exposed in vivo to doxorubicin-containing liposomes. Biochim Biophys Acta. 1988;965(2–3):136–45.CrossRefPubMedGoogle Scholar
  47. 47.
    Mayer LD, Dougherty G, Harasym TO, Bally MB. The role of tumor-associated macrophages in the delivery of liposomal doxorubicin to solid murine fibrosarcoma tumors. J Pharmacol Exp Ther. 1997;80(3):1406–14.Google Scholar
  48. 48.
    Banciu M, Schiffelers RM, Storm G. Investigation into the role of tumor-associated macrophages in the antitumor activity of doxil. Pharm Res. 2008;25(8):1948–55.CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Lília R. Cordeiro Pedrosa
    • 1
  • Timo L. M. ten Hagen
    • 1
  • Regine Süss
    • 2
  • Albert van Hell
    • 3
  • Alexander M. M. Eggermont
    • 1
    • 4
  • Marcel Verheij
    • 3
    • 5
  • Gerben A. Koning
    • 1
  1. 1.Section Surgical Oncology, Department of SurgeryLaboratory Experimental Surgical OncologyRotterdamThe Netherlands
  2. 2.Department of Pharmaceutical Technology and BiopharmacyAlbert-Ludwigs UniversityFreiburgGermany
  3. 3.Division of Biological Stress Responsehe Netherlands Cancer Institute - Antoni van Leeuwenhoek HospitalAmsterdamThe Netherlands
  4. 4.Institut de Cancerologie Gustave RoussyParisFrance
  5. 5.Department of RadiotherapyThe Netherlands Cancer Institute - Antoni van Leeuwenhoek HospitalAmsterdamThe Netherlands

Personalised recommendations