Visualization of Endosomal Escape of Intracellularly Delivered Protein With Unexpected Photochemical Internalization of Trypan Blue

ABSTRACT Intracellular protein delivery holds great potential for biomedicine and biotechnology, but real‐time monitoring of protein release remains challenging. Here, we introduce trypan blue (TB) as a multifunctional agent for both tracking and enhancing protein delivery. TB forms a supramolecular complex with GFP, quenching its fluorescence, and further assembles with a cationic guanidine‐rich polymer into a stable nanostructure. Upon 590 nm light irradiation, TB acts as a photosensitizer, triggering photochemical internalization (PCI) that promotes endosomal escape and protein release into the cytosol, accompanied by recovery of GFP fluorescence. This TB‐mediated PCI not only enhances delivery efficiency but also allows real‐time visualization of protein binding and release. We demonstrate the utility of this system through in vitro and in vivo delivery of functional proteins, including the cytotoxic protein saporin, which exhibits light‐activated antitumor activity. Our work highlights TB as a simple yet powerful tool for advancing intracellular protein delivery and tracking.