Abstract
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.
Highlights
Nanomedicine describes the use of nano-sized materials for applications including diagnostics, therapeutics and the use of nanoparticles (NPs) as drug delivery systems.[1]
In this work we present a method for the optimised production and processing of HepG2 spheroids on scale compatible with highcontent screening (HCS) microscopy
We present a high-throughput approach employing high-content screening microscopy and advanced automated image analysis to measure NP-induced cytotoxicity at multiple scales from hundreds of spheroids in parallel
Summary
Nanomedicine describes the use of nano-sized materials for applications including diagnostics, therapeutics and the use of nanoparticles (NPs) as drug delivery systems.[1] The use of NPs in this context is an attractive solution to counter several key limitations in the development of therapeutics, such as offtarget toxicity, low tissue and cell-type specificity and limited efficacy.[2] The wide range of applications associated with the emerging use of NPs in the therapeutic context is largely due to their versatility They are available in a range of sizes, shapes and materials, and they can be conjugated to therapeutics, as well as modified to bind to specific targets.
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