Poly(amidoamine)s with potential as drug carriers: degradation and cellular toxicity

Poly(amidoamine)s were synthesized by polyaddition reaction: to bis-acryloylpiperazine of piperazine (1), or N,N'-bis(2-hydroxyethyl)ethylenediamine (2), and to 2,2-bis(acrylamido)acetic acid of piperazine (3). Compound 2 was also end-capped with 4-hydroxythiophenol, thus introducing a terminal moiety suitable for radio-iodination using the chloramine T method (4). Such polymers behave as bases in aqueous solution, and their net average charge alters considerably as the pH changes from 7.4 to 5.5. This results in a change in polymer conformation which may prove useful in the design of polymeric drug delivery systems. However, their suitability for use in the organism will depend on polymer toxicity and also on their rate of biodegradation. Here we studied the biological properties of the above poly(amidoamine)s with a view to optimizing the synthesis of novel drug carriers. The general cytotoxicity of compounds 1, 2, 3, and 4 was examined in vitro using two human cell lines, hepatoma (HepG2) and a lymphoblastoid leukaemia (CCRF). Several different methods [the tetrazolium (MTT) test, [3H]leucine or [3H]thymidine incorporation, or counting cell numbers] were used to measure cell viability. Compounds 1, 2, and 4 were much less toxic to both cell lines than equivalent concentrations of the polycationic poly-L-lysine, and in no case did viability fall below 50% (concentrations up to 2 mg/ml). Although compound 2 was not markedly toxic to HepG2 cells, concentration-dependent toxicity was observed against CCRF cells. In this case, the polymer concentration decreasing viability by 59% (ID50) was approximately 50 micrograms/ml for compound 2 compared with an ID50 of approximately 10 micrograms/ml for poly-L-lysine. The rate of hydrolytic degradation of compound 2 was examined using viscometric measurements and gel permeation chromatography (GPC). After incubation at pH 7.5 and 8.0 for 24 h, polymer intrinsic viscosity was decreased by approximately 50% and GPC elution profiles showed a simultaneous increase in polymer retention time, indicating a fall in molecular weight. Hydrolytic degradation progressed much more slowly at pH 5.5. Compound 4 was also incubated with a mixture of isolated rat liver lysosomal enzymes (tritosomes) at pH 5.5, but no increase in the rate of degradation was observed.