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Macromolecular crowding and the presence of organelles in the cytosol present barriers to particle mobility, such that it is unclear how nano-carriers can deliver their active agents to the nucleus. In this work a sixth generation amino terminated polyamide polylysine dendrimer (Gly-Lys63 (NH2)64) (MW 8149, diameter 6.5 nm) which is fluorescent allowed the study of nuclear uptake and mobility in living lung carcinoma (SK/MES-1) and colon adenocarcinoma (Caco-2) cells. The dendrimer is found within 25–45 min of incubation inside the cell nuclei. Living cells were then used to develop a method for the dynamic nuclear uptake study using confocal microscopy. The dynamic uptake of the dendrimer demonstrated here allowed the apparent cytoplasmic diffusion coefficient (D) of the dendrimer to be calculated. Values were found in the range 5.99 × 10−11 cm2 s−1 (SK/MES-1 cells) to 9.82 × 10−11 cm2 s−1 (Caco-2 cells). The difference must reflect variation in the intracellular architecture of the cell types.