Quantifying the Endosomal Escape of pH-Responsive Nanoparticles Using the Split Luciferase Endosomal Escape Quantification Assay

All nanoparticles have the potential to revolutionize the delivery of therapeutic cargo such as peptides, proteins, and RNA. However, effective cytosolic delivery of cargo from nanoparticles represents a significant challenge in the design of more efficient drug delivery vehicles. Recently, research has centered on designing nanoparticles with the capacity to escape endosomes by responding to biological stimuli such as changes in pH, which occur when nanoparticles are internalized into the endo-/lysosomal pathway. Current endosomal escape assays rely on indirect measurements and yield little quantitative information, which hinders the design of more efficient drug delivery vehicles. Therefore, we adapted the highly sensitive split luciferase endosomal escape quantification (SLEEQ) assay to better understand nanoparticle-induced endosomal escape. We applied SLEEQ to evaluate the endosomal escape behavior of two pH-responsive nanoparticles: the first with a poly(2-diisopropylamino ethyl methacrylate) (PDPAEMA) core and the second with 1:1 ratio of poly(2-diethylamino ethyl methacrylate) (PDEAEMA) and PDPAEMA. SLEEQ directly measured the cytosolic delivery and showed that engineering the nanoparticle disassembly pH could improve the endosomal escape efficiency by fivefold. SLEEQ is a versatile assay that can be used for a wide range of nanomaterials and will improve the development of drug delivery vehicles in the future.