Genetically Encoded Split-Luciferase Biosensors to Measure Endosome Disruption Rapidly in Live Cells

Abstract

Endosomal escape is a critical step in intracellular delivery of biomacromolecular drugs, but quantitative, high throughput study of endosomal vesicle disruption remains elusive. We designed two genetically encoded split luciferase "turn on" reporters that can be assayed rapidly in well plates on live cells using a luminometer. Both systems use non-luminescent N-terminal and C-terminal luciferase fragments which can reconstitute a functional luminescent enzyme when they are held in proximity by their fusion partners. The first system uses Gal8 and CALCOCO2 fused to these fragments, which interact following endosome disruption and facilitate complementation of the split luciferase fragments to produce significant luminescence when luciferin is added. The second system uses the Nterminal carbohydrate recognition domain of Gal8 (G8-NCRD) fused to both luciferase fragments. Following endosome disruption, G8-NCRD binds to exposed glycans inside endosomes, concentrating both fragments there to reconstitute active luciferase. Additionally, and in contrast to recently reported Gal8 intracellular tracking with fluorescent microscopy, these split luciferase-based assays enable simultaneous identification and downselection of cytotoxic test conditions because the luciferase reaction requires intracellular ATP. Further, we demonstrate that the lead luminescent cell line is more sensitive to detection of endosomal disruption at lower doses of an endosome disrupting drug carrier than the previously reported Gal8-YFP fluorescent system. These systems represent a first-inclass luminescent assay to detect endosome disruption in high throughput while excluding toxic formulations. Endosome disruption screening with these "turn on" systems has potential as a tool in the discovery and development of intracellular biologic drug delivery formulations.

Top-Down Fabricated microPlates for Prolonged, Intra-articular Matrix Metalloproteinase 13 siRNA Nanocarrier Delivery to Reduce Post-traumatic Osteoarthritis

“…Quantification of Gal8 recruitment has been validated as a method of endosome escape analysis that is predictive of gene silencing bioactivity. 54,55 The siMMP13-μPL releasates triggered robust recruitment of Gal8-YFP puncta while direct treatment with empty μPLs did not (Figure 2E,F).…”

“…This redistribution is due to strong Gal8 binding to lectins selectively found on the inner leaflet of the endosome membrane; these lectins become exposed and available to Gal8 upon efficient disruption of the endosome. Quantification of Gal8 recruitment has been validated as a method of endosome escape analysis that is predictive of gene silencing bioactivity. , The siMMP13-μPL releasates triggered robust recruitment of Gal8-YFP puncta while direct treatment with empty μPLs did not (Figure E,F).…”

Modifying Cell Membranes with Anionic Polymer Amphiphiles Potentiates Intracellular Delivery of Cationic Peptides

“…While hemolysis is a widely used, highly reproducible assay, it does not fully recapitulate the more complex endosomal membrane composition or biochemical microenvironment. We recently reported on yellow fluorescent protein-Galectin-8 (YFP-Gal8) and split luciferase-based reporter cell lines for direct visualization and high throughput screening of endosome disruption. ,, The Gal8 machinery serves as part of the innate immune system for detection of intracellular entry by viruses and bacteria . Upon disruption of endosomal vesicles, cytosolic Gal8 binds to exposed glycans selectively localized on the inner endosomal membrane.…”

A Genetically Encoded Bioluminescence Intracellular Nanosensor for Androgen Receptor Activation Monitoring in 3D Cell Models

“…Notably, the reassembly is reversible, thus enabling both protein association and dissociation to be monitored [3]. NanoBiT has been successfully applied to monitor PPI and detect intracellular calcium levels [13], endosome disruption [14], and circulating microRNAs with such a high sensitivity that it could be implemented in smartphone-based assays [15]. In another configuration, split reporter proteins provide a single-molecule probe to monitor ligand-induced conformational changes in a single molecular backbone [16][17][18].…”