Pharmaceutical Research

, Volume 13, Issue 3, pp 352–359 | Cite as

Preparation and Characterization of Doxorubicin-Loaded Sterically Stabilized Immunoliposomes

  • Noam Emanuel
  • Eli Kedar
  • Elijah M. Bolotin
  • Nechama I. Smorodinsky
  • Yechezkel Barenholz


Purpose. To compare the performance of sterically stabilized, doxorubicin-loaded liposomes with and without surface attached specific antibodies (D-SSIL and D-SSL, respectively).

Methods. Small (≤ 120 nm) unilamellar liposomes were prepared composed of hydrogenated soy phosphatidylcholine, hydrogenated phosphatidylethanolamine (HPE), cholesterol, and 2000Da polyethylene glycol (2000PEG) attached to the primary amino group of distearoyl phosphatidylethanolamine. Doxorubicin was remote-loaded into these liposomes by an ammonium sulfate gradient to form the D-SSL. Monoclonal IgG3 NI32/2 antibodies directed against a polyoma virus tumor-associated antigen expressed on A9 etc 102 murine fibrosarcoma cells were attached to the D-SSL HPE via a thioether bond to form the D-SSIL-32/2. A control of nonspecific D-SSIL was prepared by attaching nonspecific IgG3-enriched immunoglobulins to D-SSL. All liposomes were physically and chemically characterized and then tested in vitro for tumor cell binding, specificity, and uptake by macrophages; and in vivo for the drug plasma pharmacokinetics after intravenous administration in mice.

Results. (i) The attachment of antibodies to D-SSL did not impair their chemical or physical stability and had a minimal effect on their size and level of loaded drug, (ii) The combination of specific antibodies and 2000PEG grafted in the liposomes improved the specific binding to relevant target cells by reducing the level of unspecific binding to nonrelevant cells. (iii) D-SSIL retained the prolonged circulation and slow clearance typical of SSL lacking the antibodies.

Conclusions. Sterically stabilized immunoliposomes exhibited stability, ability to recognize target cells, and prolonged circulation time. This study also shows that it is feasible to prepare them in pharmaceutically acceptable dosage form. Thus, further investigation for tumor targeting and efficacy is warranted.

sterically stabilized immunoliposomes targeting doxorubicin immunospecificity tumor cells 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    M. C. Woodle and D. D. Lasic. Sterically stabilized liposomes. Biochim. Biophys. Acta 1113:171–199 (1992).Google Scholar
  2. 2.
    L. Huang, ed. Forum: Covalently attached polymers and glycans to alter the biodistribution of liposomes. J. Liposome Res. 2:289–454 (1992).Google Scholar
  3. 3.
    A. A. Gabizon, Y. Barenholz, and M. Bialer. Prolongation of the circulation time of doxorubicin encapsulated in liposomes containing a polyethylene glycol-derivatized phospholipid: Pharmacokinetic studies in rodents and dogs. Pharm. Res. 10:703–708 (1993).Google Scholar
  4. 4.
    A. Gabizon, R. Catane, B. Uziely, B. Kaufman, T. Safra, R. Cohen, F. Martin, A. Huang, and Y. Barenholz. Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes. Cancer Res. 54:987–992 (1994).Google Scholar
  5. 5.
    D. Papahadjopoulos, T. M. Allen, A. A. Gabizon, E. Mayhew, K. 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).Google Scholar
  6. 6.
    A. H. Sehon, Suppression of antibody responses by conjugates of antigens and monomethoxypoly(ethylene glycol). Adv. Drug Delivery Rev. 6:203–217 (1991).Google Scholar
  7. 7.
    C. Delgado, G. E. Francis, and D. Fisher. The uses and properties of PEG-linked proteins. Crit. Rev. Ther. Drug Carrier Syst. 9:249–304 (1992).Google Scholar
  8. 8.
    A. L. Klibanov, K. Maruyama, A. M. Beckerleg, V. P. Torchilin, and L. Huang. Activity of amphipathic poly(ethylene glycol) 5000 to prolong the circulation time of liposomes depends on the liposome size and is unfavorable for immunoliposome binding to target. Biochim. Biophys. Acta 1062:142–148 (1991).Google Scholar
  9. 9.
    G. Blume, G. Cevc, D. J. A. Crommelin, I. A. Bakker-Woudenberg, C. Kluft, and G. Storm. Specific targeting with poly(ethylene glycol)-modified liposomes: coupling of homing devices to the ends of the polymeric chains combines effective target binding with long circulation times. Biochim. Biophys. Acta 1149:180–184 (1993).Google Scholar
  10. 10.
    G. Haran, R. Cohen, L. K. Bar, and Y. Barenholz. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. Biochim. Biophys. Acta 1151:201–215 (1993).Google Scholar
  11. 11.
    N. Emanuel, E. Kedar, E. M. Bolotin, N. I. Smorodinsky, and Y. Barenholz. Targeted delivery of doxorubicin via sterically stabilized immunoliposomes: Pharmacokinetics and biodistribution in tumor-bearing mice. Submitted for publication.Google Scholar
  12. 12.
    Y. Barenholz, and S. Amselem. Quality control assays in the development and clinical use of liposome-based formulations. In: Gregoriadis, G., ed., Liposome Technology, 2nd ed., Vol. I, CRC Press, Boca Raton, FL, 1993, pp. 527–616.Google Scholar
  13. 13.
    A. B. Langer, N. Emanuel, J. Even, W. H. Fridman, O. Gohar, B. Gonen, B. Z. Katz, M. Ran, N. I. Smorodinksy, and I. F. Witz. Phenotypic properties of 3T3 cells transformed in vitro with polyoma virus and passaged once in syngeneic animals. Immunobiology 185:281–291 (1992).Google Scholar
  14. 14.
    L. Hudson and F. C. Hay. Practical Immunology, 3rd ed., Blackwell, Oxford, 1989, pp. 316–319.Google Scholar
  15. 15.
    E. M. Bolotin, R. Cohen, L. K. Bar, N. Emanuel, S. Ninio, D. D. Lasic, and Y. Barenholz. Ammonium sulfate gradients for efficient and stable remote loading of amphipathic weak bases into liposomes and ligandoliposomes. J. Liposome Res. 4:455–479 (1994).Google Scholar
  16. 16.
    N. Emanuel, E. Kedar, O. Toker, E. Bolotin, and Y. Barenholz. Steric stabilization of liposomes improves their use in diagnostics. In: Lasic, D. D. and Barenholz, Y., eds., Handbook on Nonmedical Applications of Liposomes, Vol. IV, CRC Press, Boca Raton, FL, pp. 229–243 (1996).Google Scholar
  17. 17.
    F. J. Martin, T. D. Heath, and R. R. C. New. Covalent attachment of proteins to liposomes. In: New, R.R.C., ed., Liposomes—A Practical Approach, IRL Press, Oxford, 1990, pp. 163–182.Google Scholar
  18. 18.
    D. Goren, A. Gabizon, and Y. Barenholz. The influence of physical characteristics of liposomes containing doxorubicin on their pharmacological behavior. Biochim. Biophys. Acta 1029:285–294 (1990).Google Scholar
  19. 19.
    L. S. Minamide and J. R. Bamburg. A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents. Anal. Biochem. 190:66–70 (1990).Google Scholar
  20. 20.
    J. Cummings and C. S. McArdle. Studies on the in vivo disposition of adriamycin in human tumors which exhibit different responses to the drug. Br. J. Cancer 53:835–838 (1986).Google Scholar
  21. 21.
    L. Wish, J. Furth, and R. H. Storey. Direct determination of plasma, cell and organ-blood volumes in normal and hyperemic mice. Proc. Soc. Exp. Biol. Med. 74:644–648 (1950).Google Scholar
  22. 22.
    S. Matzku, H. Krempel, H.-P. Weckenmann, V. Schirrmacher, H. Sinn, and H. Stricker. Tumour targeting with antibody-coupled liposomes: failure to achieve accumulation in xenografts and spontaneous liver metastases. Cancer Immunol. Immunother. 31:285–291 (1990).Google Scholar
  23. 23.
    P. A. M. Peeters, C. Oussoren, W. M. C. Eling, and D. J. A. Crommelin. Unwanted interactions of maleimidophenylbutyrate-phosphatidylethanolamine containing (immuno) liposomes with cells in vitro. J. Liposome Res. 1:261–268 (1990).Google Scholar
  24. 24.
    N. C. Phillips and A. Emili. Immunogenicity of immunoliposomes. Immunol. Lett. 30:291–296 (1991).Google Scholar
  25. 25.
    S. Hashida, I. Masayoshi, S. Inoue, K.-H. Ruan, and E. Ishikawa. More useful maleimide compounds for the conjugation of Fab' to horseradish peroxidase through thiol groups in the hinge. J. Appl. Biochem. 6:56–63 (1984).Google Scholar
  26. 26.
    T. M. Allen, A. K. Agrawal, I. Ahmad, C. B. Hansen, and S. Zalipsky. Antibody-mediated targeting of long-circulating (StealthR) liposomes. J. Liposome Res. 4:1–25, 1994.Google Scholar
  27. 27.
    T. D. Madden, P. R. Harrigan, L. C. Tai, M. B. Bally, L. D. Mayer, T. E. Redelmeier, H. C. Loughrey, C. P. Tilcock, L. W. Reinish, and P. R. Cullis. The accumulation of drugs within large unilamellar vesicles exhibiting a proton gradient: a survey. Chem. Phys. Lipids 53:37–46 (1990).Google Scholar
  28. 28.
    R. Bredehorst, F. S. Ligler, A. W. Kusterbeck, E. L. Chang, B. P. Gaber, and C.-W. Vogel. Effect of covalent attachment of immunoglobulin fragments on liposomal integrity. Biochemistry 25:5693–5698 (1986).Google Scholar
  29. 29.
    I. Ahmad and T. M. Allen. Antibody-mediated specific binding and cytotoxicity of liposome-entrapped doxorubicin to lung cancer cells in vitro. Cancer Res. 52:4817–4820 (1992).Google Scholar

Copyright information

© Plenum Publishing Corporation 1996

Authors and Affiliations

  • Noam Emanuel
    • 1
  • Eli Kedar
    • 1
  • Elijah M. Bolotin
    • 2
  • Nechama I. Smorodinsky
    • 3
  • Yechezkel Barenholz
    • 2
  1. 1.The Lantenbery Center for General and Tumor ImmunologyThe Hebrew University, Hadassah Medical SchoolJerusalemIsrael
  2. 2.Department of BiochemistryThe Hebrew University, Hadassah Medical SchoolJerusalemIsrael
  3. 3.Department of Cell Research and Immunology, George S. Wise Faculty of Life SciencesTel Aviv UniversityTel AvivIsrael

Personalised recommendations