In the emerging field of nanomedicine, targeted delivery of nanoparticle encapsulated active pharmaceutical ingredients (API) is seen as a potential significant development, promising improved pharmacokinetics and reduced side effects. In this context, understanding the cellular uptake of the nanoparticles and subsequent subcellular distribution of the API is of critical importance. Doxorubicin (DOX) was encapsulated within chitosan nanoparticles to investigate its intracellular delivery in A549 cells in vitro. Unloaded (CS-TPP) and doxorubicin-loaded (DOX-CS-TPP) chitosan nanoparticles were characterised for size (473±41 nm), polydispersity index (0.3±0.2), zeta potential (34±4 mV), drug content (76±7 µM) and encapsulation efficiency (95±1%). The cytotoxic response to DOX-CS-TPP was substantially stronger than to CS-TPP, although weaker than that of the equivalent free DOX. Fluorescence microscopy showed a dissimilar pattern of distribution of DOX within the cell, being predominantly localised in the nucleus for free form and in cytoplasm for DOX-CS-TPP. Confocal microscopy demonstrated endosomal localisation of DOX-CS-TPP. Numerical simulations, based on a rate equation model to describe the uptake and distribution of the free DOX, nanoparticles and DOX loaded nanoparticles within the cells, and the subsequent dose and time dependent cytotoxic responses, were used to further elucidate the API distribution processes. The study demonstrates that encapsulation of the API in nanoparticles results in a delayed release of the drug to the cell, resulting in a delayed cellular response. This work further demonstrates the potential of mathematical modelling in combination with intracellular imaging techniques to visualise and further understand the intracellular mechanisms of action of external agents, both APIs and nanoparticles in cells
