Imaging of CPP Delivery Mechanisms of Oligonucleotides

Cationic cell-penetrating peptides spontaneously associate with negatively charged oligonucleotides to form submicron nanoparticles, so-called polyplexes. Contact with cells leads to endosomal uptake of these nanoparticles. Oligonucleotide activity critically depends on endosomal release and finally dissociation of polyplexes. Fluorescence provides a highly powerful means to follow the spatial dynamics of oligonucleotide uptake, trafficking and decomplexation, in particular when combined with markers of subcellular compartments that enable a quantitative analysis of colocalization and thereby mapping of trafficking routes. In this chapter, we describe protocols for a highly defined formation of polyplexes. We then point out the use of fluorescent fusion proteins to identify subcellular trafficking compartments and image analysis protocols to obtain quantitative information on trafficking routes and endosomal release.