This paper goes into how tracking the journey of materials following uptake by cells is critical for improving drug delivery. Many therapeutics get trapped in vesicles following entry into the cell and are unable to reach the location where they are active. Methods to examine this voyage are currently low throughput, limited by the resolution of microscopy and can miss fleeting interactions. To overcome this, the researchers developed a sensor (SNAPSwitch) that emits a signal when the material reaches a certain location in the cell such as the cytosol or nucleus. They can then look at how much material ends up at each location to get an understanding of how it travels through the cell. This information could then be used to find the delivery method that gets the most therapeutic to the right location.
Determining where material is trafficked following endocytosis is essential for understanding many cellular processes. The intracellular transport of inbound biomolecules is an integral process in cell signalling, immune responses and in the trafficking of infectious agents, such as viruses and bacterial toxins4. Furthermore, the destination of internalized material is of critical importance for the subcellular delivery of therapeutics5. Following uptake, many materials become trapped in endo/lysosomes, preventing access to their site of action. For example, nucleic acids for gene silencing or delivery require transfer to the cytosol or nucleus, respectively. To design carriers that can efficiently deliver molecules to these locations, we need to understand the trafficking of these vehicles and their ultimate subcellular fate.
SNAPSwitch, is based on benzylguanine, the native substrate for the SNAP-tag. The sensor is engineered so that when SNAPSwitch interacts with SNAP-tag, the quencher (QSY-21) is transferred to the SNAP-tag, while a fluorophore (Cy5) becomes fluorescent and remains attached to the protein/DNA/nanoparticle. After activation, the fluorescence is permanently switched on and the sensor remains attached to the material of interest. These features of SNAPSwitch offer significant advantages over typical image-based colocalization analysis and other assays for endosomal escape such as split green fluorescent protein (GFP)25. Signal from SNAPSwitch accumulates over time as more interactions occur enabling quantification of material transitioning through specific locations of the cell, such as cargo passing through an endosome into the cytosol. The fluorophore is pH insensitive within a range relevant to the endocytic pathway (pH 4–10)26, while most GFP variants are not27, which is an issue where endocytic processes are involved due to acidification endosomes. Finally, the labelled material is not anchored to the SNAP-tag after an interaction that could potentially block further trafficking. This means subsequent trafficking of the material can be observed either in the same sample or at different time points, using flow cytometry to avoid photobleaching.