The molecular dissection of the mechanisms of nanoparticle penetrance into 3D cancer cell models

Nanoparticles (NPs), which are objects ranging from 1 to 100 nm in size, have immense potential as drug delivery vehicles. In fact, nanomedicines, which refer to the combination of NPs with drugs, already represent an emerging class of therapeutic carrier, with more than 60 different nanomedicines having been approved for clinical use. The attraction towards the use of NPs in medicine comes not only from their ability to directly target diseased tissue, but also their potential for improved efficacy and safety in comparison to conventional drugs and their bioavailability. However, little mechanistic knowledge exists about how NPs interact with cells, the subcellular pathways they take, or the cellular proteins that regulate these events. To date, the overwhelming majority of studies that have assessed NP transport have been performed in 2-dimensional (2D) monolayer-grown cells. Moreover, the pathways used by different types of NPs in these cells is still highly debated, with different types of NPs utilising a variety of endocytosis pathways and trafficking to different organelles. These uptake and trafficking routes ultimately depend on factors such as the material with which the NPs are made, their size and shape, as well as their surface charge. Moreover, the type of cell used also influences the uptake and trafficking pathway utilised by NPs. It is being increasingly recognised that monolayer-grown cells are poor representative models for the study of NPs as nanomedicines, as they fail to recapitulate many of the interactions and processes that occur in tissues. Therefore, growing cells in 3D assemblies is highly favourable as they can better recapitulate in vivo conditions. The aim of this study was to develop 3D spheroid models, representing different types of tissues, that can be used to study NP uptake and transport using a high-content imaging approach. In this study, H358, HepG2 and HT-29 cells were grown as spheroids using an extracellular matrix (ECM)-derived hydrogel, namely Cultrex, which facilitates aggregation of single cells into 3D spheroids. Different cell types were grown for several days in an optical-quality 96-well imaging plate, which allowed for the generation of over 100 spheroids per well. This method enabled efficient immunostaining of spheroids and subsequent high-content confocal fluorescence imaging, thus allowing for analysis of populations of spheroids, as well as individual cells and subcellular structures such as lysosomes. Multiple z-slices were acquired for each experiment, which enabled high-content volumetric analysis of spheroids, as opposed to analysis of single representative planes. High-content analysis was also used to quantify different morphological parameters of the different spheroid models. An RNA interference (RNAi) screen was conducted in the H358 spheroid model, as these spheroids were readily transfected with small interfering RNAs (siRNAs). A total of 55 genes with known roles in trafficking through the endolysosomal system, recycling, exocytosis and degradation were downregulated in spheroids, followed by the addition of fluorescently-labelled carboxylated 40 nm polystyrene NPs for 48 hours. High-content imaging and analysis was subsequently performed. Individual spheroids were segmented and classified into four concentric layers. The fluorescence intensity of NPs within each layer was subsequently quantified. The quantitative image data was then statistically analysed and 7 genes were identified as potentially playing a role in NP uptake and trafficking in spheroids. Single cell and subcellular analysis was performed on spheroids treated with siRNAs against these candidate genes. This revealed further information about the potential mechanisms of membrane trafficking used by NPs in cells within spheroids. Taken together, this work describes a robust approach for generating 3D spheroid models that can be applied in a systematic manner to study NP transport.