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Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

A key challenge in nanomedicine stems from the continued need for a systematic understanding of the delivery of nanoparticles in live cells. Complexities in delivery are often influenced by the biophysical characteristics of nanoparticles, where even subtle changes to nanoparticle designs can alter cellular uptake, transport and activity. Close examination of these processes, especially with imaging, offers important insights that can aid in future nanoparticle design or translation. Rapid fluorescence lifetime imaging microscopy (RapidFLIM) is a potentially valuable technology for examining intracellular mechanisms of nanoparticle delivery by directly correlating visual data with changes in the biological environment. To date, applications for this technology in nanoparticle research have not been explored. A PicoQuant RapidFLIM system was used together with commercial silica nanoparticles to follow particle uptake in glioblastoma cells. Importantly, RapidFLIM imaging showed significantly improved image acquisition speeds over traditional FLIM, which enabled the tracking of nanoparticle uptake into subcellular compartments. We determined mean lifetime changes and used this to delineate significant changes in nanoparticle lifetimes (>0.39 ns), which showed clustering of these tracks proximal to both extracellular and nuclear membrane boundaries. These findings demonstrate the ability of RapidFLIM to track, localize and quantify changes in single nanoparticle fluorescence lifetimes and highlight RapidFLIM as a valuable tool for multiparameter visualization and analysis of nanoparticle molecular dynamics in live cells.

Nanoparticles hold great preclinical promise in cancer therapy but continue to suffer attrition through clinical trials. Advanced, three dimensional (3D) cellular models such as tumor spheroids can recapitulate elements of the tumor environment and are considered the superior model to evaluate nanoparticle designs. However, there is an important need to better understand nanoparticle penetration kinetics and determine how different cell characteristics may influence this nanoparticle uptake. A key challenge with current approaches for measuring nanoparticle accumulation in spheroids is that they are often static, losing spatial and temporal information which may be necessary for effective nanoparticle evaluation in 3D cell models. To overcome this challenge, we developed an analysis platform, termed the Determination of Nanoparticle Uptake in Tumor Spheroids (DONUTS), which retains spatial and temporal information during quantification, enabling evaluation of nanoparticle uptake in 3D tumor spheroids. Outperforming linear profiling methods, DONUTS was able to measure silica nanoparticle uptake to 10 μm accuracy in both isotropic and irregularly shaped cancer cell spheroids. This was then extended to determine penetration kinetics, first by a forward-in-time, center-in-space model, and then by mathematical modelling, which enabled the direct evaluation of nanoparticle penetration kinetics in different spheroid models. Nanoparticle uptake was shown to inversely relate to particle size and varied depending on the cell type, cell stiffness and density of the spheroid model. The automated analysis method we have developed can be applied to live spheroids in situ, for the advanced evaluation of nanoparticles as delivery agents in cancer therapy., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)