Monitoring endosomal escape of fluorescent cell-penetrating peptides

Cell-penetrating peptides (CPPs) have the capacity to transport cargos into cells either through endocytosis or direct translocation across the plasma membrane. CPPs entering via endocytosis remain generally trapped within endosomes and lysosomes. This entrapment can be alleviated by coupling the CPPs to endosomal escape promoting cargos or by using small molecules that permeate endosomes or lysosomes. L-Leucyl-L-Leucine methyl ester (LLOMe) is an example of such a molecule. The assessment of CPP endosomal escape into the cytosol is not trivial as cytosolic acquisition can also occur via CPP direct translocation across the plasma membrane. Moreover, visualization of CPP cytosolic acquisition often requires the CPP to be tagged with a fluorophore that can affect the behavior of the CPP and that can also be released from the CPP in the degradative environment of endosomes and lysosomes. In the present work, we have explored various parameters that can potentially affect the recording of CPP endosomal escape, such as the chirality of the amino acids making the CPPs, the nature of the fluorophore coupled to the CPPs, and the timing of the recording. For this purpose, we have used three commonly used CPPs: R9 (a synthetic CPP made of 9 arginines), TAT, and penetratin. We have used LLOMe as the endosomal escape promoter. This study presents guidelines for the assessment of CPP endosomal escape. We also provide a framework for selecting appropriate combinations of amino acid chirality, fluorophore type, and assessment timing based on what researchers want to achieve when the CPP-cargo is released from vesicles into the cytosol.