Direct cytosol delivery of mRNA by micron-sized co-assembly with designer oligopeptides

Inefficient endosomal escape has been regarded as the main bottleneck for intracellular nucleic acid delivery. While most research efforts have been spent on designing various nano-sized particles, we took a different path here, investigating micron-sized carriers for direct cytosol entry. Using the spontaneous co-assembly of mRNA and the designer 27 amino acid oligopeptide named pepMAX2, micron-sized co-assemblies were obtained with various sizes by altering the concentration of NaCl salt and time for pre-incubation. Surprisingly, transfection was much more effective using micron-sized than nano-sized co-assemblies, and the efficiency surpasses that of a widely used lipid-based commercial reagent. The study was complemented by computational simulations, inhibitor studies and live-cell confocal imaging to shed light on the role of electrostatic interaction on assembly and the mechanism of uptake and intracellular trafficking. These micron-sized co-assemblies directly enter the cytosol and then release mRNA, bypassing conventional pathways and thus avoiding the lysosomal degradation. This simple approach involving short oligopeptides and salt addition to create optimal micron-sized co-assembly with mRNA should open new avenues to overcome endosomal barriers for intracellular delivery of nucleic acid therapeutics.