Protein Nanodisk Assembling and Intracellular Trafficking Powered by An Arginine-Rich (R9) Peptide

Abstract

Arginine(R)-rich cationic peptides are powerful tools in drug delivery since, alone or when associated with polyplexes, proteins or chemicals, they confer DNA condensation, membrane translocation and blood-brain barrier crossing abilities. The unusual stability and high in vivo performance of their associated drugs suggest a particulate organization or R(n) complexes, which this study aimed to explore. We have analyzed the particulate organization and biological performance in DNA delivery of a model, R9-containing green fluorescent protein by dynamic light scattering, transmission electron microscopy, atomic force microscopy, single cell confocal microscopy and flow cytometry. A deep nanoscale examination of R9-powered constructs reveals a novel and promising feature of R9, that when fused to a scaffold green fluorescent protein, promote its efficient self-assembling as highly stable, regular disk-shaped nanoparticles of 20 x 3 nm. These constructs are efficiently internalized in mammalian cells and rapidly migrate through the cytoplasm towards the nucleus in a fully bioactive form. Besides, such particulate platforms accommodate, condense and deliver plasmid DNA to the nucleus and promote plasmid-driven transgene expression. The architectonic properties of arginine-rich peptides at the nanoscale reveal a new category of protein nanoparticles, namely nanodisks, and provide novel strategic concepts and architectonic tools for the tailored construction of new-generation artificial viruses for gene therapy and drug delivery.