Endosomal escape is a bottleneck in the efficient delivery of therapeutics using nanoparticles; therefore understanding how this property can be optimized is important for achieving better therapeutic outcomes. It has been demonstrated that pH-responsive nanoparticles (pHlexi nanoparticles) have potential to achieve effective escape from the endosomal compartments of the cell. In this paper a library of five pHlexi particles with tunable disassembly pH were synthesized by combining poly(ethylene glycol)-b-poly(2-(diethylamino)ethyl methacrylate) (PEG-b-PDEAEMA) with random copolymers of 2-(diethylamino)ethyl methacrylate and 2-(diisopropylamino)ethyl methacrylate. A series of cellular studies were conducted to investigate the effect of particle composition on in vitro behavior. Endosomal escape was probed using a calcein escape assay in NIH/3T3 fibroblast cells, demonstrating endosomal escape increased with increasing particle concentration. Interestingly, it was shown that endosomal escape was most efficient with particles that disassemble at high (pH 7.2) or low (pH 4.9) pH, with particles that disassemble between pH 5.8 and 6.6 inducing decreased levels of endosomal escape. This change in endosomal escape behavior suggests particles can induce escape by different pathways. The results show that tuning the core component of pHlexi particles can improve the effectiveness of endosomal escape capabilities and thus their ability to act as effective delivery systems.

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAPSwitch) that can be conjugated to biomolecules using click chemistry. SNAPSwitch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAPSwitch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAPSwitch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.

The internalization of proteins plays a key role in cell development, cell signaling and immunity.We have previously developed a specific hybridization internalization probe (SHIP) to quantitate the internalization of proteins and particles into cells. Herein, we extend the utility of SHIP to examine both the endocytosis and recycling of surface receptors using flow cytometry. SHIP was used to monitor endocytosis of membrane-bound transferrin receptor (TFR) and its soluble ligand transferrin (TF). SHIP enabled measurements of the proportion of surface molecules internalized, the internalization kinetics and the proportion and rate of internalized molecules that recycle to the cell surface with time. Using this method, we have demonstrated the internalization and recycling of holo-TF and an antibody against the TFR behave differently. This assay therefore highlights the implications of receptor internalization and recycling, where the internalization of the receptor-antibody complex behaves differently to the receptor-ligand complex. In addition, we observe distinct internalization patterns for these molecules expressed by different subpopulations of primary cells. SHIP provides a convenient and high throughput technique for analysis of trafficking parameters for both cell surface receptors and their ligands.

pH‐responsive nanoparticles have generated significant interest for use as drug delivery systems due to their potential for inducible release at low pH. The pH variation from the bloodstream (pH 7.4) to intracellular compartments of cells called endosomes/lysosomes (pH < 5.0) has been of particular interest. However, one of the limitations with nanoparticle delivery systems is the inability to migrate out of these compartments to the cytosol or other organelles, via a process termed endosomal escape. Previous studies have postulated that pH‐responsive nanoparticles can facilitate endosomal escape through a range of mechanisms including membrane interaction, pH‐induced swelling, and the proton‐sponge effect. In this study, a series of pH‐swellable nanoparticles (85–100 nm) are designed and their impact on biological interactions, particularly endosomal escape, are investigated. The particles exhibit tunable pH‐induced swelling (from 120% to 200%) and have good buffering capacity. The cellular association is studied using flow cytometry and endosomal escape is determined using a calcein leakage assay. Interestingly, no endosomal escape with all nanoparticle formulations is found, which suggests there are limitations with both the proton‐sponge effect and pH‐induced swelling mechanism as the primary methods for inducing endosomal escape.

Inside Front Cover: Nanoparticles which undergo pH‐induced swelling upon cell internalization are synthesized with tunable swelling and buffering capacity. Previous research has suggested such materials endosomal escape through both the proton sponge and pH‐induced swelling mechanisms. However, in this work no endosomal escape was observed, suggesting limitations with these mechanisms. This is reported by Georgina K. Such and co‐workers in article number 2100445.