Cis-aconityl spacer between daunomycin and macromolecular carriers: A model of pH-sensitive linkage releasing drug from a lysosomotropic conjugate

N-cis-Aconityl and N-maleyl derivatives of daunomycin prepared from the respective anhydrides were conjugated to Affi-Gel 701 (aminoethyl polyacrylamide beads) and to poly(D-lysine). The cis-aconityl linkage between the drug and Affi-Gel 701 is pH-sensitive with a hydrolysis half-life of less than 3 h at pH 4 and more than 96 h at pH 6 or higher. Thin-layer chromatography and cytotoxic tests in cultured cells indicate that the product of hydrolysis is unaltered daunomycin. These Affi-Gel conjugates present for 3 days in the culture medium of WEHI-5 cells at neutral pH have little or no growth inhibitory effect. N-cis-aconityl daunomycin-poly(D-lysine) conjugates, however, added to WEHI-5 cells under comparable conditions cause a 90% inhibition of cell growth. In contrast, comparable addition of N-maleyl daunomycin-poly(D-lysine) conjugates is not inhibitory. We conclude that unlike the Affi-Gel conjugate, N-cis-aconityl daunomycin-poly(D-lysine) enters cells and reaches the lysosomal compartment, and that the cis-aconityl spacer releases daunomycin from poly(D-lysine) in the acidic milieu of lysosomes due to the participation of a free cis-carboxylic group. This releasing mechanism should be applicable to other drug-macromolecular conjugates.