(476c) Heterotrifunctional Scaffolds for Measuring Intracellular Bond Cleavage Kinetics

Cleavable bonds have long been an attractive intracellular switch for nanoparticle and conjugate-based therapeutics. A variety of cleavable bonds have been designed to respond to intracellular stimuli such as pH gradients, reducing environments, and enzymatic activity. However, the rationale behind choosing an optimal cleavable chemistry has remained elusive. To establish design rules, we have sought to quantify the kinetics of sub-cellular bond cleavage in a cell type and endocytic pathway dependent manner. Towards this end, we have developed a novel, cleavable heterotrifunctional chemical cross-linker capable of incorporating targeting ligands and environmentally responsive bonds in a modular fashion. This sequence-defined, oligothioetheramide-based platform addresses many of the synthetic and functional limitations of traditional cleavable, heteromultifunctional chemical cross-linkers.

In a proof-of-principle study, we synthesized four heterotrifunctional oligoTEA cross-linkers with up to two orthogonal internal cleavage sites¹. To demonstrate their utility as multifunctional probes, these linkers were conjugated to a pair of fluorophores that undergo Förster resonance energy transfer (FRET) and a model protein - human transferrin. Confocal microscopy of these transferrin probes showed differential fluorescence emission intensities, indicating disulfide bond reduction within the endocytic transferrin pathway. To quantify the kinetics of this cleavage event, a quenched fluorescence probe was analyzed via quantitative live-cell imaging in three different cell lines. The data presented herein will inform the design of a variety of drug delivery platforms fine-tuned for cell-specific and pathway-specific release of active drug cargo.

1. Sorkin, M. R., Walker, J. A., Brown, J. S. & Alabi, C. A. Versatile Platform for the Synthesis of Orthogonally Cleavable Heteromultifunctional Cross-Linkers. Bioconjugate Chemistry (2017). doi:10.1021/acs.bioconjchem.7b00033