Development of a new method to detect the entry of label-free peptides into vesicle lumen

      Madhabi et al have published the paper entitled with “Detection of the entry of nonlabeled transportan 10 into single vesicles” (Biochemistry, 59, 1780-1790 (2020)) [Abstract]. To detect the entry of cell-penetrating peptides (CPPs) into the lumen of lipid vesicles such as giant unilamellar vesicles (GUVs) and the cytoplasm of cells, fluorescent probe-labeled CPPs have been used. In this paper, we have developed a new method to detect the entry of label-free (or nonlabeled) CPPs into the lumen of single GUVs. In this method, GUVs contain large unilamellar vesicles (LUVs) whose lumen contains a high concentration of fluorescent probe, calcein. The fluorescence intensity (FI) of the GUV lumen due to calcein is small due to the self-quenching of calcein fluorescence. If CPPs enter the GUV lumen and induce pore formation in the LUV’s membrane, calcein leaks from the LUV lumen to the GUV lumen, which enhances the FI of the GUV lumen. The lipid compositions of LUVs are selected to allow leakage from the LUVs but not from the GUVs during their interactions with CPPs. Using this method, we examined the interaction of label-free CPP, transportan 10 (TP10) with single GUVs containing AF647 and LUVs mentioned above using the single GUV method with confocal laser scanning microscopy. After starting the interaction of TP10 with single GUVs, the FI of the GUV lumen due to calcein increased without changing the FI due to AG647 as the interaction time of TP10 increased. This result indicates the entry of label-free TP10 into single GUV lumen without pore formation.