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Publication
Gal8 Visualization of Endosome Disruption Predicts Carrier-Mediated Biologic
Drug Intracellular Bioavailability.
Authors Kilchrist KV, Dimobi SC, Jackson MA, Evans BC, Werfel TA, Dailing EA,
Bedingfield SK, Kelly IB, Duvall CL
Submitted By Submitted Externally on 2/12/2020
Status Published
Journal ACS nano
Year 2019
Date Published 2/1/2019
Volume : Pages 13 : 1136 - 1152
PubMed Reference 30629431
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 < 10-4) 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.




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