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

Endosomal escape is a critical step in the intracellular delivery of biomacromolecular drugs, but a quantitative, high-throughput study of endosomal-vesicle disruption remains elusive. We designed two genetically encoded split-luciferase turn-on reporter assays that can be measured rapidly in well plates on live cells using a luminometer. Both systems use nonluminescent N-terminal and C-terminal luciferase fragments that can reconstitute a functional luminescent enzyme when they are colocalized by their fusion partners. The first system uses luciferase-fragment fusion to Galectin 8 (Gal8) and CALCOCO2. Gal8 and CALCOCO2 interact following endosomal-vesicle disruption to facilitate luciferase complementation into the active enzyme, enabling a luminescence readout (G8C2 system). The second system expresses the N-terminal carbohydrate recognition domain (N-CRD) of Gal8 fused to each luciferase fragment (G8G8 system). Following endosome disruption, G8-NCRD binds to exposed glycans inside endosomes, concentrating both fragments in close proximity and reconstituting active luciferase. The G8G8 system emerged as the lead reporter candidate and was further characterized by comparing it to previously reported Gal8-YFP tracking using microscopy. We also characterized the G8G8 system response to several commercial and research drug-delivery reagents: DOTAP lipid, JetPEI, Lipofectamine 2000, and a library of polymers with known endosomal-escape activity, revealing dose-dependent increases in luminescence due to endosomal disruption. These new reporters provide a first-in-class luminescent assay to rapidly detect endosome disruption in a high-throughput format while excluding toxic formulations. Endosome-disruption screening with these turn-on assays has the potential to accelerate and to improve the rigor of programs focused on the discovery and development of intracellular biologic drug-delivery formulations.

Polymeric biomaterials for biophotonic applications.

With the growing importance of optical techniques in medical diagnosis and treatment, there exists a pressing need to develop and optimize materials platform for biophotonic applications. Particularly, the design of biocompatible and biodegradable materials with desired optical, mechanical, chemical, and biological properties is required to enable clinically relevant biophotonic devices for translating in vitro optical techniques into in situ and in vivo use. This technological trend propels the development of natural and synthetic polymeric biomaterials to replace traditional brittle, nondegradable silica glass based optical materials. In this review, we present an overview of the advances in polymeric optical material development, optical device design and fabrication techniques, and the accompanying applications to imaging, sensing and phototherapy.