Mechanistic studies of in vitro cytotoxicity of PAMAM dendrimers in mammalian cells

The in vitro cytotoxic response of human dermal and colon cell lines to structurally well defined full generation cationic dendritic polyamidoamine (PAMAM) nanoparticles was investigated. Dendrimers of generations G4, G5, G6 were chosen for this study. PAMAM dendrimers have been demonstrated to elicit a well defined cytotoxicological response from Alamar Blue, Neutral Red and MTT assays, where the response increases systematically with dendrimer generation and number of surface amino groups. A good correlation was found between the EC₅₀ values of these assays. This systematic response is furthermore demonstrated for the generation of reactive oxygen species, mitochondrial membrane potential, inflammatory responses, lysosomal activity, cell cycle interference, caspase activation, onset of apoptosis and levels of DNA damage. The mechanism of endosomal escape of PAMAM by the so-called 'proton-sponge effect' was also studied. The results are consistent with a pathway of the endosomal uptake of PAMAM, followed by the endosomal rupture and subsequent localisation of PAMAM dendrimers in the mitochondria, leading to PAMAM generation, dose and time dependant biphasic ROS production, mitochondrial membrane potential decay and caspase- 8 and 3 activation, inflammatory responses (TNF-α, IL-6 and IL-8 expression), apoptosis and DNA damage (by TUNEL assay). Overall, significant differences are observed between the responses of the dermal and colon cell lines, and it is suggested that these can be understood in terms of differing intrinsic antioxidant levels.