Unraveling the Mechanisms of Peptide-Mediated Delivery of Nucleic Acids Using Electron Microscopy

Abstract

Cell-penetrating peptides (CPPs) are efficient non-viral delivery vectors for bioactive cargos, both in vitro and in vivo. Cargo molecules can be attached to CPPs either via covalent conjugation or by complex formation using co-incubation, which is typically used for charged molecules such as nucleic acids. The latter technique is efficiently used in case of CADY, MPG, Pep peptides, NickFects and PepFects that condense oligonucleotides (ONs) into nanoparticles, which efficiently enter cells and induce biological effects. Despite being highly promising candidates for developing new-generation medicines, CPPs' internalization mechanisms and intracellular trafficking are still far from being well-understood, and obtained data are often controversial. Transmission electron microscopy (TEM) is an informative and valuable tool for examining the mechanisms of CPP-ON nanoparticles. TEM enables to visualize nanoparticles or single molecules labeled with Nanogold™ tag, and follow their association with cells and intracellular localization. In this chapter, we present methods for preparation of CPP-ON nanoparticles for TEM analysis and for examination of their interactions with the plasma membrane, and subsequent cellular uptake either by direct translocation or endocytosis. In case of endocytosis, ONs have to be released from endosomes and reach their target site in nucleus or cytoplasm to reveal their activity. TEM enables to estimate when the endosomal escape begins, from which type of endosomal vesicles it occurs, whether the vesicles are broken, or nanocomplexes translocate across the membrane into cytosol. Since single ONs could be followed, the time-frame that is necessary for the splice-switching nucleotides to translocate into cell nucleus can be analyzed by TEM.

Role of autophagy in cell-penetrating peptide transfection model

“…Maturation and acidification of endosomal vesicles 34 are necessary for the cargo molecules to escape from a particular subset of endosomes. In analogy with our report 35 and earlier studies, after 4 h of uptake, endosomes with the discontinuous limiting membrane (Fig. 6e,f , arrowheads) appeared in cells, and the complexes escaped into the cytosol, which is a very rare event per se 36 .…”

Studies of cell-penetrating peptides by biophysical methods

“…Alternatively, a label can also be put on cargo. This has been used with oligonucleotide cargoes that were usually labelled with colloidal gold particles or quantum dots (Arukuusk et al ., 2013; Margus et al ., 2015). Very rarely were TEM observations used as the single detection method, but was frequently combined with other biophysical and biochemical methods.…”

Characteristics of Cell-Penetrating Peptide/Nucleic Acid Nanoparticles

“…For experiments with SCO-NG the thiol group at 5′ end of SCO was tagged with nanogold cluster (Monomaleimido Nanogold, Nanoprobes, NY, U.S.A., d = 1.4 nm) by forming a covalent bond between the thiol group of oligonucleotide and the maleimide group of label as described earlier. 42 CPP-SCO-NG complexes were formed as described above.…”