Nanoparticle drug delivery systems have significant potential to transform precision medicine due to their ability to encapsulate a wide range of cargo, improve systemic circulation time, and enhance targeted delivery. These delivery properties can be further modified by altering the surface chemistry of the nanoparticle carrier. However, a gap remains in our understanding of the cellular mechanisms underlying nanoparticle uptake, in addition to the subcellular trafficking kinetics. A comprehensive understanding of nanoparticle-cell interactions considering both the nanocarrier and the drug cargo has been challenging, in part due to technological limitations. Here, we present a robust imaging workflow to study long-term dynamics of nanoparticle delivery in live cells. We show that integration of holography and tomography enhances the study of live cells in a labelfree environment and can be combined with intermittent fluorescence microscopy to assess nanoparticle uptake and delivery kinetics for up to 30 hours. We also describe a method to quantitatively characterize uptake of a library of fluorescently tagged lipid-and polymer-based nanoformulations without introducing cell or organelle markers. This application of dynamic fluorescent holotomography as a novel method to investigate nanoparticle uptake and cargo delivery highlights the expanding utility of multimodal, label-free, live imaging techniques.
