Mechanobiology

Gal8 Visualization of Endosome Disruption Predicts Carrier-Mediated Biologic Drug Intracellular Bioavailability

AUTHORS

ABSTRACT

Endolysosome entrapment is one of the key barriers to the therapeutic use of biologic drugs that act intracellularly. The screening of prospective nanoscale endosome-disrupting delivery technologies is currently limited by methods that are indirect and cumbersome. Here, we statistically validate Galectin 8 (Gal8) intracellular tracking as a superior approach that is direct, quantitative, and predictive of therapeutic cargo intracellular bioactivity through in vitro high-throughput screening and in vivo validation. Gal8 is a cytosolically dispersed protein that, when endosomes are disrupted, redistributes by binding to glycosylation moieties selectively located on the inner face of endosomal membranes. The quantitative redistribution of a Gal8 fluorescent fusion protein from the cytosol into endosomes is demonstrated as a real-time, live-cell assessment of endosomal integrity that does not require labeling or modification of either the carrier or the biologic drug and that allows quantitative distinction between closely related, endosome-disruptive drug carriers. Through screening two families of siRNA polymeric carrier compositions at varying dosages, we show that Gal8 endosomal recruitment correlates strongly ( r = 0.95 and p < 10) with intracellular siRNA bioactivity. Through this screen, we gathered insights into how composition and molecular weight affect endosome disruption activity of poly[(ethylene glycol)- b-[(2-(dimethylamino)ethyl methacrylate)- co-(butyl methacrylate)]] [PEG-(DMAEMA- co-BMA)] siRNA delivery systems. Additional studies showed that Gal8 recruitment predicts intracellular bioactivity better than current standard methods such as Lysotracker colocalization ( r = 0.35, not significant), pH-dependent hemolysis (not significant), or cellular uptake ( r = 0.73 and p < 10). Importantly, the Gal8 recruitment method is also amenable to fully objective high-throughput screening using automated image acquisition and quantitative image analysis, with a robust estimated Z’ of 0.6 (whereas assays with Z’ > 0 have high-throughput screening utility). Finally, we also provide measurements of in vivo endosomal disruption based on Gal8 visualization ( p < 0.03) of a nanocarrier formulation confirmed to produce significant cytosolic delivery and bioactivity of siRNA within tumors ( p < 0.02). In sum, this report establishes the utility of Gal8 subcellular tracking for the rapid optimization and high-throughput screening of the endosome disruption potency of intracellular delivery technologies.