Unravelling the endosomal escape of pH-responsive nanoparticles using the split luciferase endosomal escape quantification assay

Endosomal escape is a major bottleneck for efficient intracellular delivery of therapeutic cargoes, particularly for macromolecular biological cargoes such as peptides, proteins and nucleic acids. pH-responsive polymeric nanoparticles that can respond to changes in the pH of intracellular microenvironments have generated substantial interest in navigating the endosomal barrier. In this study, we applied the highly sensitive split luciferase endosomal escape quantification (SLEEQ) assay to better understand the endosomal escape efficiency of dual component pH-responsive nanoparticles based on poly(2-(diethylamino) ethyl methacrylate) (PDEAEMA) and poly(2-(diisopropylamino) ethyl methacrylate) (PDPAEMA). Previous work investigated the use of a disulfide-linked HiBiT peptide conjugate encapsulated within the nanoparticle core, which upon meeting the LgBiT protein in the cytosol demonstrated luminescence which could be quantified to assess endosomal escape. However, we were interested in understanding whether this assay could be tuned to understand the endosomal escape of both a therapeutic cargo and a larger carrier. To achieve this, we designed two different HiBiT conjugates by applying a carbonylacrylic-functionalized thioether (non-cleavable) linker, which is more stable in endosomes, and a less stable disulfide (cleavable) linker to attach HiBiT to the nanoparticle core. Nanoparticles with disulfide-linked HiBiT demonstrated a higher endosomal escape efficiency of 6-7%, whereas thioether-linked HiBiT demonstrated <3% endosomal escape efficiency with a twofold decrease in cytosolic delivery. This suggests that degradation of the disulfide linker in endosomes leads to cytosolic delivery of a free HiBiT cargo, while thioether-linked HiBiT polymers are larger and thus fewer HiBiT-carrier conjugates can escape the endosomes. Overall, this work demonstrates that the SLEEQ assay can be tuned to understand the cytosolic delivery of different components based on the use of different linker chemistries and thus it is an important tool for designing therapeutic delivery systems in the future.