The role of endocytosis in the uptake and intracellular trafficking of PepFect14–nucleic acid nanocomplexes via class A scavenger receptors

Cell penetrating peptides are efficient tools to deliver various bioactive cargos into cells, but their exact functioning mechanism is still debated. Recently, we showed that a delivery peptide PepFect14 condenses oligonucleotides (ON) into negatively charged nanocomplexes that are taken up by cells via class A scavenger receptors (SR-As). Here we unraveled the uptake mechanism and intracellular trafficking of PF14–ON nanocomplexes in HeLa cells. Macropinocytosis and caveolae-mediated endocytosis are responsible for the intracellular functionality of nucleic acids packed into nanocomplexes. However, only a negligible fraction of the complexes were trafficked to endoplasmic reticulum or Golgi apparatus — the common destinations of caveolar endocytosis. Neither were the PF14–SCO nanocomplexes routed to endo-lysosomal pathway, and they stayed in vesicles with slightly acidic pH, which were not marked with LysoSensor. “Naked” ON, in contrary, was rapidly targeted to acidic vesicles and lysosomes. The transmission electron microscopy analysis of interactions between SR-As and PF14–ON nanocomplexes on ultrastructural level revealed that nanocomplexes localized on the plasma membrane in close proximity to SR-As and their colocalization is retained in cells, suggesting that PF14–ON complexes associate with targeted receptors.