A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery

Abstract

RNA-based therapies have great potential to treat many undruggable human diseases. However, their efficacy, in particular for mRNA, remains hampered by poor cellular delivery and limited endosomal escape. Advances in the development and rational optimisation of delivery vectors, such as lipid nanoparticles (LNPs), are impeded by the limited availability of screening methods that probe the intracellular processing of LNPs in sufficient detail. We have developed a high-throughput imaging-based endosomal escape assay utilising a Galectin-9 reporter and fluorescently labelled mRNA to probe correlations between nanoparticle-mediated uptake, endosomal escape frequency, and mRNA translation. This assay has, furthermore, been integrated with a screening platform for nanoparticle formulation to optimise LNPs. We show that Galectin-9 recruitment is a robust, quantitative reporter of endosomal escape events induced by different mRNA delivery nanoparticles and small molecules. We also identify nanoparticles with superior escape properties and demonstrate significant cell line variances in endosomal escape response, highlighting the need for fine-tuning of delivery formulations for specific applications.

Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides

“…Next, we sought to ensure that the increase in SSO activity was linked to endosomal rupture. Galectin-9 (GAL9) is a known marker of endosomal membrane disruption and can be utilized to quantitate endosomal rupture 22 . The initially selected 7 compounds and all 14 analogs of CMP05 were subjected to an mCherry-GAL9-recruitment assay to screen for the ability to induce endosomal rupture in both HeLa and HuH7 cell lines.…”

“…The levels of splice switching correlate to observations obtained from quantitative analyses of the endosomal structure utilizing super-resolution fluorescence microscopy. We then demonstrate how functional SSO delivery occurs in a time and concentration-dependent manner correlating with endosomal rupture in a previously established microscopic GAL9 based assay 22 . Furthermore, we demonstrate the use of live-cell SSO-endosomal tracking microscopy to quantitate the colocalization of SSOs to various endosomal compartments over time.…”

“…The field has acknowledged the need for widely applicable, sensitive characterization strategies to explore and optimize the use of SMCs for functional ON delivery, with novel imaging modalities at the forefront of this development 19 . For example, recent work has utilized live-cell microscopic assays which visualizes the recruitment of galectin-8 and galectin-9 (GAL8 and GAL9, respectively) to damaged endosomal membranes, and they have further been able to quantitate the galectin response in the presence of CADs [20][21][22] . In addition, the ability to visualize the mechanisms driving ON escape from endosomes by utilizing super-resolution microscopy is proving integral to understanding how SMCs work in the context of ON delivery 23 .…”

How can we use the endocytosis pathways to design nanoparticle drug-delivery vehicles to target cancer cells over healthy cells?

“…185 In addition, endosomal and lysosomal escape kinetics of nanoparticles have cell line-specific differences. 186,187 These differences might arise because the endosomal compartments are different between cell types or it might be because of other differences in the protein corona formed that may change nanoparticle fusion and endosomal escape properties. 186 Also, the position and the size of intracellular compartments (endosome, lysosome) within the cell determines the pH of the compartments and the escape strategy for the internalized nanoparticles.…”

“…186,187 These differences might arise because the endosomal compartments are different between cell types or it might be because of other differences in the protein corona formed that may change nanoparticle fusion and endosomal escape properties. 186 Also, the position and the size of intracellular compartments (endosome, lysosome) within the cell determines the pH of the compartments and the escape strategy for the internalized nanoparticles. 182,188,189 Lysosomes located near the cell membranes were found to be less acidic than the lysosomes located near the nucleus.…”