Temporal Microenvironment Mapping (μMap) of Intracellular Trafficking Pathways of Cell-Penetrating Peptides Across the Blood-Brain Barrier

Peptides play critical roles in cellular functions such as signaling and immune regulation, and peptide-based biotherapeutics show great promise for treating various diseases. Among these, cell-penetrating peptides (CPPs) are particularly valuable for drug delivery due to their ability to cross cell membranes. However, the mechanisms underlying CPP-mediated transport, especially across the blood-brain barrier (BBB), remain poorly understood. Mapping intracellular CPP pathways is essential for advancing drug delivery systems, particularly for neurological disorders, as understanding how CPPs navigate the complex environment of the BBB could enable the development of more effective brain-targeted therapies. Here, we leverage a nanoscale proximity labeling technique, termed µMap, to precisely probe the peptide– receptor interactions and intracellular trafficking mechanisms of photocatalyst-tagged CPPs. The unique advantage of the μMap platform lies in the ability to control the timing of light exposure, which enables the collection of time-gated data, depending on when the blue light is applied to the cells. By harnessing this spatiotemporal precision, we can uncover key peptide–receptor interactions and cellular processes, setting the stage for new innovations in drug design and brain-targeted therapies.