Cell-penetrating peptide-based systems for nucleic acid delivery: a biological and biophysical approach

Abstract

The increasing knowledge on the genetic basis of disease provides a platform for the development of promising gene-targeted therapies that can be applied to numerous pathological conditions, including cancer. Such genetic-based approaches involve the use of nucleic acids as therapeutic agents, either for the insertion or for the repair and regulation of specific genes. However, despite the huge pharmacological potential of these molecules, their application remains highly dependent on the development of delivery systems capable of mediating efficient cellular uptake. The discovery of a class of small peptides, the so-called cell-penetrating peptides (CPPs), which are able to very efficiently cross cell membranes through a mechanism that is independent of membrane receptors or transporters and avoids lysosomal enzymatic degradation, has been enthusiastically considered of key interest to improve noninvasive cellular delivery of therapeutic molecules. A large number of CPPs have been applied successfully to mediate the intracellular delivery of nucleic acids, including the S4(13)PV peptide for which interactions with membranes and resulting biological effects are illustrated in this chapter. Here, we provide a description of the experimental procedures for the preparation of CPP-based nucleic acid complexes and assessment of their formation, the selection of those protocols leading to the most efficient complexes, the biophysical characterization of CPP membrane interactions, and the evaluation of the biological and cytotoxic activity of the complexes.