Resolving subcellular pH with a quantitative fluorescent lifetime biosensor.

Changes in sub-cellular pH play a key role in metabolism, membrane transport, and triggering cargo release from therapeutic delivery systems. Most methods to measure pH rely on intensity changes of pH sensitive fluorophores, however, these measurements are hampered by high uncertainty in the inferred pH and the need for multiple fluorophores. To address this, here we combine pH dependant fluorescent lifetime imaging microscopy (pHLIM) with deep learning to accurately quantify sub-cellular pH in individual vesicles. We engineer the pH sensitive protein mApple to localise in the cytosol, endosomes, and lysosomes, and demonstrate that pHLIM can rapidly detect pH changes induced by drugs such as bafilomycin A1 and chloroquine. We also demonstrate that polyethylenimine (a common transfection reagent) does not exhibit a proton sponge effect and had no measurable impact on the pH of endocytic vesicles. pHLIM is a simple and quantitative method that will help to understand drug action and disease progression.

Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay.

Cytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.

Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics.

Endocytosis is a critical step in the process by which many therapeutic nanomedicines reach their intracellular targets. Our understanding of cellular uptake mechanisms has developed substantially in the past five years. However, these advances in cell biology have not fully translated to the nanoscience and therapeutics literature. Misconceptions surrounding the role of different endocytic pathways and how to study these pathways are hindering progress in developing improved nanoparticle therapies. Here, we summarize the latest insights into cellular uptake mechanisms and pathways. We highlight limitations of current systems to study endocytosis, particularly problems with non-specific inhibitors. We also summarize alternative genetic approaches to robustly probe these pathways and discuss the need to understand how cells endocytose particles in vivo. We hope that this critical assessment of the current methods used in studying nanoparticle uptake will guide future studies at the interface of cell biology and nanomedicine.

A molecular sensor to quantify the localization of proteins, DNA and nanoparticles in cells.

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.

Controlling endosomal escape using nanoparticle composition: current progress and future perspectives.

Polymer nanoparticles offer significant benefits for improving delivery of biological therapeutics such as DNA and proteins, as they allow the cargo to be protected until it is delivered to a target cell. However, there are still challenges with achieving efficient delivery to the optimal cellular region. One significant roadblock is escape of nanoparticles from within the endosomal/lysosomal compartments into the cytosol. Here, we review the recent advances in understanding endosomal escape of polymer nanoparticles. We also discuss the current progress on investigating how nanoparticle structure can control endosomal escape. It is important to understand the fundamental biological processes that govern endosomal escape in order to design more effective therapeutic delivery systems.