CPP-protein constructs induce a population of non-acidic vesicles during trafficking through endo-lysosomal pathway

The major limitation in the application of bioactive molecules is their low permeation across plasma membrane. Effective transporters — cell-penetrating peptides (CPPs) — are utilized to enhance uptake of various cargo upon attachment to its sequences. Still, information about relevance of different endocytic routes during CPP–cargo internalization is ambiguous and underlying mechanism(s) of intracellular trafficking is even less understood. We first defined involvement of recycling pathway in trafficking of 3 different CPPs — transportan, oligoarginine and Tat — complexed to avidin-TexasRed in Cos-7 cells in relation to trans-Golgi network spatially constraining recycling endosomes. By confocal microscopy, only a negligible fraction of complexes-containing vesicles were found inside trans-Golgi ring suggesting its marginal role in CPP-mediated delivery. Secondly, we characterized engagement of endo-lysosomal pathway to assess acidity of complexes-containing vesicles. CPPs induced 3 different populations of complexes-containing vesicles which size and proportion depended on CPP, time and concentration. In time, more complexes were targeted to low-pH structures. However, a population of complexes-containing vesicles was observed to retain rather neutral pH. Induction of vesicles with non-acidic pH generated i.e. by caveolin-dependent endocytosis or by CPPs themselves during intracellular trafficking could be the key step in inducement of escape of complexes from endosomal structures, a limiting step in effective cargo delivery by CPPs.