Quantifying nanotherapeutics penetration using hydrogel based microsystem as a new 3D in-vitro platform

Despite progress in therapeutic strategies and understanding of cancer cell biology, there is a large attrition of promising therapeutics into the clinic. One predominant reason is the huge gap between 2D in-vitro assays used for drug screening, and the in-vivo 3D-physiological environment. This is particularly important for a specific category of emerging therapeutics: nanoparticles. The lack of physiological context hampered reliable predictions for the route and accumulation of those nanoparticles in-vivo. For such nanotherapeutics, Multi-Cellular Tumour Spheroids (MCTS) is emerging as a good alternative in-vitro model. However, the classical approaches to produce MCTS suffer from low yield, poor reproducibility and slow process, while spheroid-on-chip set-ups developed so far require a microfluidic practical knowledge difficult to transfer to a cell biology laboratory. We present here a simple yet highly flexible 3D-model microsystem consisting of agarose-based micro-wells. Fully compatible with the multi-well plates format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. It is compatible with live high-resolution optical microscopy and provides a user-friendly platform for in-situ immunostaining. As a proof-of-principle of the relevance of such in-vitro platform for the evaluation of nanoparticles, the aim of this study was to analyse the kinetic and localization of nanoparticles within colorectal cancer cells (HCT-116) MCTS. The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI and confocal microscopy (AGuIX(R), currently implicated in clinical trials as effective radiosensitizers for radiotherapy). We show that the amount of AGuIX(R) nanoparticles within cells is largely different in 2D and 3D. Using our flexible agarose-based microsystems, we are able to resolve spatially and temporally the penetration and distribution of AGuIX(R) nanoparticles within tumour spheroids. The nanoparticles are first found in both extracellular and intracellular space of spheroids, within lysosomes compartment. While the extracellular part is washed away after few days, we evidenced trafficking of AGuIX(R) nanoparticles that are also found within mitochondria. Our agarose-based microsystem appears hence as a promising 3D in-vitro platform for investigation of nanotherapeutics transport, ahead of in-vivo studies.