The synthesis and evaluation of some polyamine conjugates in drug delivery.

This study investigated polyamines conjugated to either the nitrogen mustard chlorambucil, or to a fluorescent analogue (MANT). De novo syntheses were used where full control over regioselectivity was required, but derivatisation of naturally occuring polyamines, exploiting the regioselectivity of the BOC protecting group, was used where possible. The structures of two key compounds spermidine-chlorambucil (1) and spermidine-MANT (2), are shown. The reaction kinetics of chlorambucil and spermidine-chlorambucil were investigated in buffered aqueous solution and found to be first order under all conditions. Both compounds were subject to a common ion effect in the presence of chloride. Their rates of reaction were pH independent over the pH range 8 to 3 .5, but dropped rapidly below pH 3.5, corresponding to the pKa's of the aryl amine groups. The rates of reaction of both compounds were shown to be sensitive to the polarity of the medium, with similar decreases in rate in 50% aqueous acetone, and in solutions of the non-ionic surfactant Triton X-100. In the presence of charged surfactants, the compounds showed different rates of reaction, implying a specificity of interaction with surfactant head groups. Interactions with DNA were studied by fluorescence quenching and DNA crosslinking assays. The results suggest that conjugates interact with DNA similarly to free polyamines as binding is primarily electrostatic and dependent on polyamine charge. Transport across cell membranes and intracellular location of spermidine-MANT was demonstrated by fluorescence microscopy. Uptake was i) polyamine-mediated, ii) inhibited by the addition of spermidine, iii) tolerant of structural modification, and iv) saturatable, indicating an active mechanism. The fluorescent conjugate was located within cytoplasmic vesicles, and co-localised with lucifer yellow, which enters endosomes. These results show polyamine conjugates enter cells via an active polyamine transporter, possibly by receptor-mediated endocytosis.