Amphipathic helical peptide-Nile Red probes for fluorescence probing of the lipid packing defects and their surrounding membranes on exosomes

Amphipathic helical (AH) peptide-based fluorescent probes were explored for analysis of lipid packing defects (LPDs) in the membrane surface of exosomes. Two kinds of AH peptide sequences, derived from the C-terminal sequence of Apolipoprotein A-I (ApoC) and from human α-synuclein (p2-23), were examined, where they differ in the hydrophobic face that can be inserted into LPDs. From the examination of the insertion depth of the AH peptides and the competitive binding using synthetic liposomes as exosome models, we found that ApoC peptide could serve as a binder for deep LPDs whereas p2-23 peptide preferentially recognize shallow LPDs. These peptides conjugated with an environment-sensitive dye Nile Red (NR) were demonstrated to be useful for assessing both the abundance of target LPDs by the fluorescent enhancement response and the membrane properties surrounding these LPDs by the emission wavelength of the probes, respectively. With these properties, our probes successfully characterized the LPDs of exosomes from three kinds of cancer cells (A549, Hela and MCF7 cells). We showed that exosomal membranes exhibited unique structural properties regarding deep and shallow LPDs and their surrounding membrane polarity. In addition, these properties significantly depended on the donor cells. Our probes would serve as powerful tools for LPD analysis with a view toward a better understanding of exosomal membranes.