Preparation of pH-sensitive, long-circulating and EGFR-targeted immunoliposomes


A long-circulating formulation of pH-sensitive liposomes (PSLs) with antibodies against epidermal growth factor receptor (EGFR) attached was designed, expecting an increase in binding and delivery of liposomes to the target cells including non-small cell lung cancer (NSCLC) cells. Physicochemical properties of the PSLs were measured by SEM and DLS. Leakage of a self-quenching fluorescent probe, calcein, from the liposome was studied for the evaluation of pH-sensitivity. Encapsulation efficiency of gemcitabine (an anti-cancer drug) in PSLs was about 67%. Average size of liposomes was 88 nm in diameter. The PSL of DOPE/CHEMS (6:4 molar ratio) formulation showed a dramatic pH-sensitivity at/around pH 5.5, whereas non-PSL of DPPC/Chol or PC/CHEMS formulation did not. Anti-proliferation effect of gemcitabine-encapsulating PSLs & Ab-PSLs in A549 cells was 2-fold higher than the free drug, which was further elucidated by the apoptosis of the cells by gemcitabine (∼10% apoptosis for PSL or Ab-PSL formulation vs. ∼1% for free drug or non-PSL formulation) using FACS analysis. These data demonstrate delivery of gemcitabine to tumor cells can be improved by long-circulating PSLs or Ab-PSLs formulations in vitro.

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  1. Ahmad, I. and Allen, T. M., Antibody-mediated specific binding and cytotoxicity of liposome-entrapped doxorubicin to lung cancer cells in vitro, Cancer Res., 52, 4817 (1992).

    PubMed  CAS  Google Scholar 

  2. Allen, T. M., Austin, G. A., Chonn, A., Lin, L., and Lee, K. C., Uptake of liposomes by cultured mouse bone marrow macrophages: influence of liposome composition and size, Biochim. Biophys. Acta., 1061, 56–64 (1991).

    PubMed  Article  CAS  Google Scholar 

  3. Allen, T. M., Brandeis, E., Hansen, C. B., Kao, G. Y., and Zalipsky, S., A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells, Biochim. Biophys. Acta., 1237, 99 (1995).

    PubMed  Article  Google Scholar 

  4. Alving, C. R., Liposomes: Biophysics to Therapeutics. Marcel Dekker, New York, (1987).

    Google Scholar 

  5. Bangham, A. D., Standish, M. M., and Watkins, J. C., Diffusion of univalent ions across the lamellae of swollen phospholipids, J. Mol. Biol., 13, 238–252 (1965).

    PubMed  CAS  Article  Google Scholar 

  6. Bartlett, G. R., Phosphorus assay in column chromatography, J. Biol. Chem., 234, 466–468 (1959).

    PubMed  CAS  Google Scholar 

  7. Bligh, E. G. and Dyer, W. J., A rapid method of total lipid extraction and purification, Can. J. Med. Sci., 37, 911–917 (1959).

    CAS  Google Scholar 

  8. Bradford, M. M., A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248–254 (1976).

    PubMed  Article  CAS  Google Scholar 

  9. Carlsson, J., Drevin, H., and Axen, R., Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent, Biochem. J., 173, 723 (1978).

    PubMed  CAS  Google Scholar 

  10. Debs, R. J., Heath, T. D., and Papahadjopuolos, D., Targeting of anti-Thy 1.1 monoclonal antibody conjugated liposomes in Thy 1.1 mice after intravenous administration, Biochim. Biophys. Acta., 901, 183 (1987).

    PubMed  Article  CAS  Google Scholar 

  11. Heath, T. D. and Lopez, N. G., 5-Fluoroorotate: a new liposome-dependent cytotoxic agent, FEBS Lett., 187, 73–75 (1985).

    PubMed  Article  CAS  Google Scholar 

  12. Heath, T. D., Montgomery, J. A., Piper, J. R., and Papahadjopuolos, D., Antibody-targeted liposomes: increase in specific toxicity of methotrexate-gamma-aspartate, Proc. Natl. Acad. Sci. USA., 80, 1377 (1983).

    PubMed  Article  CAS  Google Scholar 

  13. Martin, F. J., Heath, T. D., and New, R. R. C., Liposomes: A practical approach, IRL press, Oxford, (1990).

    Google Scholar 

  14. Peschka-Suss, R. and Schubert, R., Liposomes. Oxford University Press, New York, (2003).

    Google Scholar 

  15. Slepushkin, V. A., Simões, S., Dazin, P., Newman, M. S., Guo, L. S., Lima, M. C., and Düzgüneş, N., Sterically stabilized pH-sensitive liposomes. Intracellular delivery of aqueous contents and prolonged circulation in vivo, J. Biol. Chem., 272, 2382–2388 (1997).

    PubMed  Article  CAS  Google Scholar 

  16. Szoka, F. and Papahadjopoulos D., Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation, Proc. Natl. Acad. Sci. U.S.A., 75, 4194–4198 (1978).

    PubMed  Article  CAS  Google Scholar 

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Correspondence to Jin-Seok Kim.

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Kim, MJ., Lee, H.J., Lee, IA. et al. Preparation of pH-sensitive, long-circulating and EGFR-targeted immunoliposomes. Arch. Pharm. Res. 31, 539 (2008).

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Key words

  • pH-Sensitive liposome (PSLs)
  • Gemcitabine
  • EGFR
  • Targeting