Multiparametric nanoparticle-induced toxicity readouts with single cell resolution in HepG2 multicellular tumour spheroids.

The use of nanomaterials as therapeutic delivery vehicles requires their careful pre-clinical evaluation. Of particular importance in this regard is measurement of cellular toxicity, ideally assessing multiple parameters in parallel from various relevant subcellular organelles. In recent years it has become evident that in vitro monolayer-grown cells do not always accurately predict any toxicity response seen in vivo, and so there is a need for more sophisticated in vitro cell models, employing a greater depth of characterisation. In this work we present an automated high-content screening microscopy approach for quantifying nanoparticle-induced toxicity in a three-dimensional multicellular tumour spheroid (MCTS) cell model. As a proof-of-principle, we perform a comparative toxicity profile study of carboxylate- versus amine-modified polystyrene nanoparticles in HepG2 spheroids. Following treatment with these nanoparticle types, we demonstrate that several hundred spheroids, of various sizes, can be morphologically profiled in a single well using automated high-content image analysis. This provides a first level of information about spheroid health in response to nanoparticle treatment. Using a range of fluorescent reporters assessing membrane permeability, lysosome function and mitochondrial activity, we also show that nanoparticle-induced toxicity information can be obtained from individual cells with subcellular resolution. Strikingly, our work demonstrates that individual cells do not all behave in a consistent manner within a spheroid structure after exposure to nanoparticles. This highlights the need for toxicity studies to not only assess an appropriate number of spheroids, but also the importance of extracting information at the subcellular level.

Investigation of the Cellular Destination of Fluorescently Labeled Carbon Nanohorns in Cultured Cells.

The high surface area, facile functionalization, and biocompatibility of carbon nanohorns (CNHs) make them attractive for many applications, including drug delivery. The cellular destination of nanomaterials dictates both the therapeutic application and the potential toxicity. Identifying the uptake mechanism is challenging as several endocytic pathways have been identified that facilitate cellular entry. Here, the cellular uptake of fluorescently labeled CNHs was assessed by utilizing quantitative cell-based assays to determine the factors influencing how internalization occurs and the destinations they reach in HeLa cells. Cell viability assays suggest that about 80% of the cells remained viable even at the highest concentration of 20 µg/mL exposure to CNHs. Uptake studies revealed that when pulse-chase conditions were applied, CNHs were seen to be localized both at the cell periphery and in a juxtanuclear pattern inside HeLa cells, in the latter case colocalizing with the lysosomal marker LAMP1. RNA interference studies, using a panel of RNA tools to individually deplete key molecules associated with the endocytic machinery, failed to block the internalization of CNHs into cells, suggesting that multiple mechanisms of endocytosis are used by this particle type.