Interaction of liposomes bearing a lipophilic doxorubicin prodrug with tumor cells

  • N. R. Kuznetsova
  • E. V. Svirshchevskaya
  • I. V. Skripnik
  • E. N. Zarudnaya
  • A. N. Benke
  • G. P. Gaenko
  • Yu. G. Molotkovskii
  • E. L. Vodovozova
Articles

Abstract

When used as nanosized carriers, liposomes enable targeted delivery and decrease systemic toxicity of antitumor agents significantly. However, slow unloading of liposomes inside cells diminishes the treatment efficiency. The problem could be overcome by the adoption of lipophilic prodrugs tailored for incorporation into lipid bilayer of liposomes. We prepared liposomes of egg yolk phosphatidylcholine and yeast phosphatidylinositol bearing a diglyceride conjugate of an antitumor antibiotic doxorubicin (a lipophilic prodrug, DOX-DG) in the membrane to study how these formulations interact with tumor cells. We also prepared liposomes of rigid bilayer-forming lipids, such as a mixture of dipalmitoylphosphatidylcholine and cholesterol, bearing DOX in the inner water volume, both pegylated (with polyethylene glycol (PEG) chains exposed to water phase) and non-pegylated. Efficiency of binding of free and liposomal doxorubicin with tumor cells was evaluated in vitro using spectrofluorimetry of cell extracts and flow cytometry. Intracellular traffic of the formulations was investigated by confocal microscopy; co-localization of DOX fluorescence with organelle trackers was estimated. All liposomal formulations of DOX were shown to distribute to organelles retarding its transport to nucleus. Intracellular distribution of liposomal DOX depended on liposome structure and pegylation. We conclude that the most probable mechanism of the lipophilic prodrug penetration into a cell is liposome-mediated endosomal pathway.

Keywords

liposomes drug delivery doxorubicin lipophilic prodrugs cell organelles intracellular traffic 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Fenske D.B., Cullis P.R. 2008. Liposomal nanomedicines. Expert Opin. Drug Deliv. 5, 25–44.PubMedCrossRefGoogle Scholar
  2. 2.
    Yuan F., Dellian M., Fukumura D., Leunig M., Berk D.A., Torchilin V.P., Jain R.K. 1995. Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res. 55, 3752–3756.PubMedGoogle Scholar
  3. 3.
    Gabizon A., Schmeeda H., Barenholz Y. 2003. Pharmacokinetics of pegylated liposomal doxorubicin: A review of animal and human studies. Clin. Pharmacokinet. 42, 419–436.PubMedCrossRefGoogle Scholar
  4. 4.
    Lasic D.D., Papahadjopoulos D. 1995. Liposomes revisited. Science. 267, 1275–1276.PubMedCrossRefGoogle Scholar
  5. 5.
    Zucker D., Marcus D., Barenholz Y., Goldblum A. 2009. Liposome drugs’ loading efficiency: A working model based on loading conditions and drug’s physicochemical properties. J. Control Release. 139, 73–80.PubMedCrossRefGoogle Scholar
  6. 6.
    Allen T.M., Cullis P.R. 2004. Drug delivery systems: Entering the mainstream. Science. 303, 1818–1822.PubMedCrossRefGoogle Scholar
  7. 7.
    Vodovozova E.L., Kuznetsova N.R., Kadykov V.A., Khutsyan S.S., Gaenko G.P., Molotkovskii J.G. 2008. Liposomes as nanocarriers of lipid-conjugated antitumor drugs melphalan and methotrexate. Ros. nanotekhonologii (Rus.). 3(3–4), 162–172 [Transl. version in Nanotechnologies in Russia. 3 (3–4), 228–239].Google Scholar
  8. 8.
    Kuznetsova N., Kandyba A., Vostrov I., Kadykov V., Gaenko G., Molotkovsky J., Vodovozova E. 2009. Liposomes loaded with lipophilic prodrugs of methotrexate and melphalan as convenient drug delivery vehicles. J. Drug Deliv. Sci. Technol. 19, 51–59.Google Scholar
  9. 9.
    Kozlov A.M., Korchagina E.Yu., Vodovozova E.L., Bovin N.V., Molotkovskii Yu.G., Syrkin A.B. 1997. Enhancement of antitumor activity of sarcolysin by its transformation to lipid derivative and incorporation into membrane of liposomes equipped with a carbohydrate targeting ligand. Bull. eksperim. biol. med. (Rus.). 123, 439–441.CrossRefGoogle Scholar
  10. 10.
    Vodovozova E.L., Moiseeva E.V., Gayenko G.P., Nifant’ev N.E., Bovin N.V., Molotkovsky J.G. 2000. Antitumor activity of cytotoxic liposomes equipped with selectin ligand SiaLex in mouse mammary adenocarcinoma. Eur. J. Cancer. 36, 942–947.PubMedCrossRefGoogle Scholar
  11. 11.
    Vodovozova E.L., Moiseeva E.V., Gaenko G.P., Bovin N.V., Molotkovskii Yu.G. 2008. Application of lipidconjugated chemotherapeutics in liposomes as a method to enhance anticancer effect of the drugs. Ros. bioterapevt. zhurn. (Rus.). 7(2), 24–33.Google Scholar
  12. 12.
    Tsuruta W., Tsurushima H., Yamamoto T., Suzuki K., Yamazaki N., Matsumura A. 2009. Application of liposomes incorporating doxorubicin with sialyl Lewis X to prevent stenosis after rat carotid artery injury. Biomaterials. 30, 118–125.PubMedCrossRefGoogle Scholar
  13. 13.
    Gabizon A., Shiota R., Papahadjopoulos D. 1989. Pharmacokinetics and tissue distribution of doxorubicin encapsulated in stable liposomes with long circulation times. J. Natl. Cancer Inst. (Bethesda). 81, 1484–1488.CrossRefGoogle Scholar
  14. 14.
    Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods. 65, 55–63.PubMedCrossRefGoogle Scholar
  15. 15.
    Mayer L.D., Hope M.J., Cullis P.R. 1986. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim. Biophys. Acta. 858, 161–168.PubMedCrossRefGoogle Scholar
  16. 16.
    Funaki N.O., Tanaka J., Kohmoto M., Sugiyama T., Ohshio G., Nonaka A., Yotsumoto F., Takeda Y., Imamura M. 2001. Membrane fluidity correlates with liver cancer cell proliferation and infiltration potential. Oncol. Rep. 8, 527–532.PubMedGoogle Scholar
  17. 17.
    Gabizon A., Papahadjopoulos D. 1988. Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc. Natl. Acad. Sci. USA. 85, 6949–6953.PubMedCrossRefGoogle Scholar
  18. 18.
    Moghimi S.M., Andersen A.J., Hashemi S.H., Lettiero B., Ahmadvand D., Hunter A.C., Andresen T.L., Hamad I., Szebeni J. 2010. Complement activation cascade triggered by PEG#PL engineered nanomedicines and carbon nanotubes: The challenges ahead. J. Control. Release. 146, 175–181.PubMedCrossRefGoogle Scholar
  19. 19.
    Karukstis K.K., Thompson E.H.Z., Whiles J.A., Rosenfeld R.J. 1998. Deciphering the fluorescence signature of daunomycin and doxorubicin. Biophys. Chem. 73, 249–263.PubMedCrossRefGoogle Scholar
  20. 20.
    Menezes D.E.L., Kirchmeier M.J., Gagne J.F., Pilarski L.M., Allen T.M. 1999. Cellular trafficking and cytotoxicity of anti-CD-targeted liposomal doxorubicin in B lymphoma cells. J. Liposome Res. 9, 199–228.CrossRefGoogle Scholar
  21. 21.
    Roepe P.D. 1992. Analysis of the steady-state and initial rate of doxorubicin efflux from a series of multidrugresistant cells expressing different levels of P-glycoprotein. Biochemistry. 31, 12555–12564.PubMedCrossRefGoogle Scholar
  22. 22.
    Cressman S., Dobson I., Lee J.B., Tam Y.Y.C., Cullis P.R. 2009. Synthesis of a labeled RGD-lipid, its incorporation into liposomal nanoparticles, and their trafficking in cultured endothelial cells. Bioconjugate Chem. 20, 1404–1411.CrossRefGoogle Scholar
  23. 23.
    Theodossiou T.A., Galanou M.C., Paleos C.M. 2008. Novel amiodarone-doxorubicin cocktail liposomes enhance doxorubicin retention and cytotoxicity in DU145 human prostate carcinoma cells. J. Med. Chem. 51, 6067–6074.PubMedCrossRefGoogle Scholar
  24. 24.
    Swift L.P., Rephaeli A., Nudelman A., Phillips D.R., Cutts S.M. 2006. Doxorubicin-DNA adducts induce a non-topoisomerase II-mediated form of cell death. Cancer Res. 66, 987–992.CrossRefGoogle Scholar
  25. 25.
    Gigli M., Doglia S.M., Millot J.M., Valentini L., Manfait M. 1988. Quantitalive study of doxorubicin in living cell nuclei by microspectrofluorometry. Biochim. Biophys. Acta. 950, 13–20.PubMedCrossRefGoogle Scholar
  26. 26.
    Fiallo M., Laigle A., Borrel M.N., Garnier-Suillerot A. 1993. Accumulation of degradation products of doxorubicin and pirarubicin formed in cell culture medium within sensitive and resistant cells. Biochem. Pharmacol. 45, 659–665.PubMedCrossRefGoogle Scholar
  27. 27.
    Hovorka O., Subr V., Vetvicka D., Kovar L., Strohalm J., Strohalm M., Benda A., Hof M., Ulbrich K., Rihova B. 2010. Spectral analysis of doxorubicin accumulation and the indirect quantification of its DNA intercalation. Eur. J. Pharm. Biopharm. 76, 514–524.PubMedCrossRefGoogle Scholar
  28. 28.
    Johannes L., Lamaze C. 2002. Clathrin-dependent or not: Is it still the question? Traffic. 3, 443–451.PubMedCrossRefGoogle Scholar
  29. 29.
    Yi X., Zimmerman M.C., Yang R., Tong J., Vinogradov S., Kabanov A.V. 2010. Pluronic-modified superoxide dismutase 1 attenuates angiotensin II-induced increase in intracellular superoxide in neurons. Free Radic. Biol. Med. 49, 548–558.PubMedCrossRefGoogle Scholar
  30. 30.
    Raggers R.J., Pomorski T., Holthuis J.C.M., Kälin N., van Meer G. 2000. Lipid traffic: The ABC of transbilayer movement. Traffic. 1, 226–234.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2013

Authors and Affiliations

  • N. R. Kuznetsova
    • 1
  • E. V. Svirshchevskaya
    • 1
  • I. V. Skripnik
    • 1
  • E. N. Zarudnaya
    • 1
  • A. N. Benke
    • 1
  • G. P. Gaenko
    • 1
  • Yu. G. Molotkovskii
    • 1
  • E. L. Vodovozova
    • 1
  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryMoscowRussia

Personalised recommendations