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

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 (Ga18) 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(-4)) 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(-3)). 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.