title: 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.
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, 23a Riia Street, Tartu, 51010, Estonia.
authors.forename: Helerin
authors.initials: H
authors.lastname: Margus
authors.forename: Carmen
authors.initials: C
authors.lastname: Juks
authors.forename: Margus
authors.initials: M
authors.lastname: Pooga
mesh_terms: D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D004705:Endocytosis; D006046:Gold / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D053758:Nanoparticles / Q000737:chemistry / Q000378:metabolism*; D009696:Nucleic Acids / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D009841:Oligonucleotides / Q000008:administration & dosage* / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D016610:Tissue Embedding / Q000379:methods
chemical_list: D057846:Cell-Penetrating Peptides; D009696:Nucleic Acids; D009841:Oligonucleotides; D006046:Gold