A high content screening microscopy approach to dissect nanoparticle uptake and trafficking in mammalian cells

Synthetic nanoparticles (NPs) are promising tools for imaging and drug delivery; however their mechanisms of cellular internalisation and trafficking await full characterisation. Current knowledge suggests that following endocytosis many types of uncoated NPs get delivered into endosomes and lysosomes. However, the mechanisms of cellular internalisation and trafficking of nanomaterials depend on a variety of factors, including the structural and physicochemical characteristics of the material itself, the biological molecules coating the nanomaterial (the corona), as well as specific cell-type differences in the endocytic machinery. In order to design effective drug delivery strategies that can use or by-pass the endocytic pathway, a comprehensive understanding of NP uptake mechanisms is therefore necessary. The present study describes design, testing, optimisation and application of an RNA interference (RNAi)-based high content screening (HCS) microscopy strategy to assess the intracellular trafficking of fluorescently-labelled 40 nm negatively charged polystyrene carboxylated NPs in HeLa cells.Firstly, NPs were characterised and a NP uptake assay and HCS analysis routine to evaluate internalisation and trafficking of NPs was developed. The assay was first implemented for a set of 10 siRNAs targeting known components of various endocytic mechanisms. A role for the large GTPase dynamin2 in trafficking of NPs to LAMP1-positive compartments was determined by automated HCS, thus establishing the principle that cellular depletion of individual endocytosis components is sufficient to result in a phenotypic effect in terms of delivery of NPs to endo/lysosomes.Secondly, a library targeting 58 proteins belonging to the family of Rab small GTPases was interrogated. siRNA treatments inducing a strong reduction of NP trafficking to LAMP1-positive membranes were validated by both HCS and quantitative PCR analysis. This revealed a role for the late endosomal RAB7A in the process, as well as identifying a previously unreported role for the Golgi associated RAB33B. Further relevance of RAB33B was investigated using a GFP-tagged protein overexpression approach, revealing a significant role for RAB33B and its GTPase activating protein (GAP) OATL1 in the trafficking of NPs through early endocytic membranes and late endosomes/lysosomes.This approach was next extended to an siRNA library targeting 348 genes involved in cytoskeleton organisation, function and regulation. A total of 39 siRNA treatments resulted in a strong decrease in NP trafficking. Of particular note was the identification of several motor protein subunits (DYNC1H1, KIF15, and MYO6) and proteins associated with actin and microtubule structure and remodelling (ARPC2, CDC42, CFL1, PLS3, TUBB). Among these, the motor protein myosin VI (MYO6), a recently established regulator of endocytosis and autophagy was selected for further studies in the context of GFP-tagged protein overexpression. This revealed partial co-localisation of NPs with myosin VI positive structures, and relevance of its cargo-selective autophagy receptor interacting motif for the trafficking of NPs to LAMP1-positive membranes.Finally, the RNAi HCS approach was used to systematically deplete 21,585 genes annotated in the human genome as a first attempt to comprehensively map the main regulators of internalisation and trafficking of NPs. In silico validation of the strongest candidate genes was carried out in the context of previously published transcriptomic data. Annotation, clustering and enrichment analyses of the validated target genes were also carried out. Bioinformatic protein-protein interaction studies of enriched target gene clusters annotated as being associated with the endomembrane system were performed, further providing evidence of the involvement of clathrin coated vesicle components (CLTC, DNM2 and AP2) and the cytoskeleton (ARPC2) in the intracellular trafficking of NPs. These studies also revealed potential roles for several endosomal proteins (RABEPK, RABEP1, HOPS/CORVET), TGN to endosomal trafficking complexes (COGs, TRAPPs, AP1) and lysosomal acidification machinery (V-ATP6).In conclusion, the results presented in this work provide the first comprehensive molecular overview of the bio-nano interaction space paving the way for the design of improved intracellular drug delivery strategies.