Comparison of cellular uptake using 22 CPPs in 4 different cell lines

Cell-penetrating peptides (CPPs) are short peptides able to penetrate cell membranes and translocate different cargoes into cells. Although recently the topic of many research articles, to our best knowledge no single systematic study of CPPs has been carried out as yet, meaning information can only by gathered piece by piece from different sources. We therefore decided to start analytical screening of CPP specificity in cell lines. We used 22 different CPPs, which have all been published before, and present the first analytical screen in 4 selected cell lines (MDCK, HEK293, HeLa, and Cos-7). Furthermore, we examined the influence of different conditions, such as protease inhibitors, incubation conditions, endocytosis inhibitors, temperature, and cytotoxicity. We clearly demonstrate that the 22 CPPs can be classified into 3 groups based on their internalization properties, even after trypsinization. Moreover, we show that additional agents, which should increase cellular uptake or dissolve endosomal/lysosomal entrapped CPPs, only have low effects. Our intensive screening under standardized conditions provides the opportunity to compare cellular uptake of CPPs, an important step for the use of CPPs as peptidic vectors in the medical field.