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Cytotoxicity of Diphtheria Toxin A Fragment to Toxin-resistant Murine Cells Delivered by pH-sensitive Immunoliposomes¹

Department of Biochemistry, University of Tennessee, Knoxville, Tennessee 37996-0840

pH-sensitive immunoliposomes composed of dioleoylphosphatidyl-ethanolamine and oleic acid (8:2 molar ratio) mediated the delivery of the cytotoxic fragment A of diphtheria toxin to the cytoplasm of target L-929 cells. Free fragment A, fragment A encapsulated in antibody-free liposomes, or fragment A encapsulated in pH-insensitive immunoliposomes was not effective in the inhibition of the cellular protein synthesis. pH-sensitive immunoliposomes containing diphtheria fragment A were not toxic to nontarget diphtheria-resistant A31 cells or to nontarget diphtheria-sensitive Vero cells. Pretreatment of target L-929 cells with the weak bases NH₄Cl or chloroquine, agents which raise the endosome/lysosome pH, blocked the cytotoxic effect of the pH-sensitive immunoliposomes containing fragment A. Excess free antibody or excess empty pH-sensitive immunoliposomes also blocked the cytotoxic effect. Since it is known that fragment A alone cannot cross lipid membranes, the results of this study indicate that pH-sensitive immunoliposomes are able to release the toxin into the cytoplasm, probably by fusing with the endosome membrane following a receptor-mediated endocytosis of the immunoliposome.

¹ This investigation was funded by NIH Grant CA 24553.

² Research Career Development Awardee (CA 00718). To whom requests for reprints should be addressed, at University of Tennessee, Department of Biochemistry, M407 Walters Life Science Building, Knoxville, TN 37996-0840.

Received 8/27/86. Revised 10/24/86. Accepted 10/27/86.