Abstract
Purpose. To investigate differences in the cellular uptake and intracellular distribution of protein-bound doxorubicin in comparison to free doxorubicin and a liposomal formulation (CAELYX®)
Methods. LXFL 529 lung carcinoma cells were incubated with an acid-sensitive transferrin and albumin conjugate of doxorubicin, a stable albumin doxorubicin conjugate, and free and liposomal doxorubicin for up to 24 h. The uptake of doxorubicin was detected with confocal laser scanning microscopy (CLSM). To investigate the intracellular localization of the anticancer drug, lysosomes, Golgi apparatus, and mitochondria were also stained by various organelle-specific fluorescent markers. In vitro efficacy of the doxorubicin derivatives was examined with the BrdU incorporation assay.
Results. The acid-sensitive albumin and transferrin doxorubicin conjugates showed enhanced cytotoxicity in comparison to liposomal doxorubicin, whereas the stable albumin-doxorubicin conjugate showed only marginal activity. Of all compounds tested, doxorubicin showed the highest cytotoxicity. CLSM studies with specific markers for lysosomes, mitochondria, and the Golgi apparatus demonstrated that protein-bound doxorubicin or liberated doxorubicin was accumulated in the mitochondria and Golgi compartments, but not in the lysosomes after 24 h. Free doxorubicin showed a time-dependent intracellular shift from the nucleus to the mitochondria and Golgi apparatus. Fluorescence resulting from incubation with CAELYX was primarily detected in the nucleus.
Conclusions. Our results indicate that other organelles in addition to the cell nucleus are important sites of accumulation and interaction for protein-bound doxorubicin or intracellularly released doxorubicin as well as for free doxorubicin.
Similar content being viewed by others
REFERENCES
Y. Takakura and M. Hashida. Macromolecular drug carrier systems in cancer chemotherapy: macromolecular prodrugs. Crit. Rev. Oncol. Hematol. 18:207-231 (1994).
G. Gregoriadis (ed.). Liposomes as Drug Carriers: Recent Trends and Progress, John Wiley, Chichester, 1988.
H. Maeda and Y. Matsumura. Tumoritropic and lymphotropic principles of macromolecular prodrugs. Crit. Rev. Ther. Drug Carrier Syst. 6:193-210 (1989).
F. Kratz, U. Beyer, T. Roth, N. Tarasova, P. Collery, F. Lechenault, A. Cazabat, P. Schumacher, C. Unger, and U. Falken. Transferrin conjugates of doxorubicin: Synthesis, characterization, cellular uptake, and in vitro efficacy. J. Pharm. Sci. 87:338-346 (1998).
F. Kratz, U. Beyer, P. Collery, F. Lechenault, A. Cazabat, P. Schumacher, U. Falken, and C. Unger. Preparation, characterization, and in vitro efficacy of albumin conjugates of doxorubicin. Biol. Pharm. Bull. 21:56-61 (1998).
F. Kratz, U. Beyer, and M. T. Schütte. Drug-polymer conjugates containing acid-cleavable bonds, Crit. Rev. Ther. Drug Carrier Syst. 16:245-288 (1999).
J. Drevs, I. Hofmann, D. Marmé, C. Unger, and F. Kratz. In vivo and in vitro efficacy of an acid-sensitive albumin conjugate of adriamycin compared to the parent compound in murine renal cell carcinoma. Drug Deliv. 6:1-7 (1999).
F. Kratz, T. Roth, I. Fichtner, H. H. Fiebig, and C. Unger: Development of antitumor protein and polymer conjugates using in vitro and in vivo human tumor xenograft models. In H. H. Fiebig and A. M. Burger (eds.), Contributions to Oncology, Vol. 54, Relevance of Tumor Models for Anticancer Drug Development, 1999, pp. 389-395.
T. M. Allen. Liposomes. Opportunities in drug delivery. Drugs 54:8-14 (1997).
A. Gordon, J. Fleagle, D. Guthrie, D. Parrkin, M. Gore, A. Lacave, and D. Mutch. Interim analysis of a phase II randomized trial of Doxil/Caelyx (D) versus topotecan (T) in the treatment of patients with relapsed ovarian cancer. Proc. Am. Soc. Oncol. 19:380a (2000).
D. Shotton and N. White. Confocal scanning microscopy: three dimensional biological imaging. Trends Biochem. Sci. 14:435-439 (1989).
R. C. Gonzales and P. Wintz. Digital Image Processing, Addison-Wesley, Reading, MA, 1987.
B. Rothen-Rutishauser, S. D. Krämer, A. Braun, M. Günthert, and H. Wunderli-Allenspach. MDCK cell cultures as an epithelial in vitro model: Cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy. Pharm. Res. 15:964-971 (1998).
F. Kratz, R. Müller-Driver, I. Hofmann, J. Drevs, and C. Unger. A novel macromolecular prodrug concept exploiting endogenous serum albumin as a drug carrier for cancer chemotherapy. J. Med. Chem. 43:1253-1256 (2000).
P. A. Rodrigues, P. Schumacher, U. Beyer, T. Roth, H. H. Fiebig, C. Unger, D. Paper, L. Messori, P. Orioli, R. Mülhaupt, and F. Kratz. Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and cellular uptake. Bioorg. Med. Chem. 7:2517-2524 (1999).
H. M. Coley, W. B. Amoa, P. R. Twentyman, and P. Workman. Examination by laser scanning confocal fluorescence imaging microscopy of the subcellular localisation of anthracyclines in parent and multidrug resistant cell lines. Br. J. Cancer 67:1316-1323 (1993).
A. Krishan, K. S. Sridhar, E. Davilla, C. Vogel, and W. Sternheim. Patterns of anthracycline retention modulation in human tumor cells. Cytometry 8:306-314 (1987).
K. K. Karukstis, E. H. Z. Thompson, J. A. Whiles, and R. J. Rosenfeld. Deciphering the fluorescence of daunomycin and doxorubicin. Biophys. Chem. 73:249-263 (1998).
J. B. Chaires, N. Dattagupta, and D. M. Crothers. Studies on the interaction of anthracycline antibiotics and deoxyribonucleic acid: Equilibrium binding studies on interaction of daunomycin with deoxyribonucleic acid. Biochemistry 21:3933-3940 (1982).
K. Barabas, J. A. Sizensky, and W. P. Faulk. Transferrin conjugates of adriamycin are cytotoxic without intercalating nuclear DNA. J. Biol. Chem. 267:9437-9442 (1992).
D. E. Lopes de Menezes, M. J. Kirchmeister, J. Gagne, L. M. Pilarski, and T. M. Allen. Cellular trafficking and cytotoxicity of anti-CD19-targeted liposomal doxorubicin in B lymphoma cells, J. Liposome Res. 9:199-228 (1999).
S. Mukherjee, R. N. Grosh, and F. R. Maxfield. Endocytosis. Physiol. Rev. 77:759-803 (1997).
K. Nicolay and B. De Kruijff. Effects of adriamycin on respiratory chain activities in mitochondria from rat liver, rat heart and bovine heart. Evidence for preferential inhibition of complex III and IV. Biochim. Biophys. Acta 892:320-330 (1987).
K. Nicolay, J. J. Fok, W. Voorhout, J. A. Post, and B. De Kruijff. Cytofluorescence detection of adriamycin-mitochondria interactions in isolated, perfused rat heart. Biochim. Biophys. Acta 887:35-41 (1986).
U. Beyer, T. Roth, A. Unold, P. Schumacher, G. Maier, A. W. Frahm, H. H. Fiebig, C. Unger, and F. Kratz. Transferrin conjugates of chlorambucil: Synthesis, in vitro efficacy and structure-activity-relationship. J. Med. Chem. 41:2701-2708 (1998).
F. Kratz, U. Beyer, T. Roth, M. T. Schütte, A. Unold., H. H. Fiebig, and C. Unger. Albumin conjugates of the anticancer drug chlorambucil: Synthesis, characterization and in vitro efficacy. Arch. Pharm., Pharm. Med. Chem. 331:47-53 (1998).
Brock, J. H. Transferrins. In P. M. Harrison (ed.), Metalloproteins, Part 2: Metal Proteins with Non-redox Roles, Verlag Chemie, Weinheim, 1985, pp. 183-263.
T. M. Allen, E. Brandeis, C. B. Hansen, G. Y. Kao, and S. Zalipsky. A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells. Biochim. Biophys. Acta 123:99-108 (1995).
D. Papahadjopoulos, T. M. Allen, A. Gabizon, A. Mayhew, E. Matthay, S. K. Huang, K. D. Lee, M. C. Woodle, D. D. Lasic, C. Redemann, and F. J. Martin. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc. Natl. Acad. Sci. USA 88:11460-11464 (1991).
N. Z. Wu, R. D. Braun, M. H. Gaber, G. M. Lim, E. T. Ong, S. Shan, D. Papahadjopoulos, and W. Dewhirst. Simultaneous measurement of liposome extravasation and content release in tumors. Microcirculation 4:83-101 (1997).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Beyer, U., Rothen-Rutishauser, B., Unger, C. et al. Differences in the Intracellular Distribution of Acid-Sensitive Doxorubicin-Protein Conjugates in Comparison to Free and Liposomal Formulated Doxorubicin as Shown by Confocal Microscopy. Pharm Res 18, 29–38 (2001). https://doi.org/10.1023/A:1011018525121
Issue Date:
DOI: https://doi.org/10.1023/A:1011018525121